Departments of Virology1 and Pediatrics2, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
Author for correspondence: Masao Yamada. Fax +81 86 235 7169. e-mail masao{at}med.okayama-u.ac.jp
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Abstract |
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Introduction |
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Several studies have reported an association of thrombocytopenia with exanthema subitum (Kitamura et al., 1994 ; Nishimura & Igarashi, 1977
; Saijo et al., 1995
; Yoshikawa et al., 1991
, 1998
). Recently, it has been reported that HHV-6 is associated with delayed engraftment after stem cell transplantation (Kadakia et al., 1996
; Maeda et al., 1999
). However, it is not clear how HHV-6 causes thrombocytopenia in primary infection or under immunosuppressive conditions. In a previous report, we showed that HHV-6 suppresses colony formation of granulocyte/macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) in vitro, indicating that HHV-6 has suppressive effects on the haematopoiesis of granulocyte/macrophage and erythroid lineages (Isomura et al., 1997
). In this study, we set out to evaluate the suppressive effects of HHV-6 and HHV-7 on the haematopoiesis of the megakaryocytic lineage by establishing culture conditions for megakaryocyte colony-forming units (CFU-Meg) in a semi-solid matrix.
Previously, it had been difficult to establish conditions suitable for CFU-Meg colony assays. Recently, however, several groups reported the isolation and cloning of human thrombopoietin (TPO), a c-mpl ligand (Bartley et al., 1994 ; de Sauvage et al., 1994
; Kaushansky et al., 1994
; Wendling et al., 1994
). TPO is a cytokine that stimulates megakaryocytic colonies and increases the number of megakaryocytic progenitors (Hagiwara et al., 1998
; Hogge et al., 1997
). We took advantage of the availability of recombinant human TPO to establish culture conditions for CFU-Meg and examined the effects of HHV-6 and HHV-7 on CFU-Meg colonies.
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Methods |
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T-cell depletion from CBMNCs.
T cells were depleted from CBMNCs by rosette formation with neuraminidase-treated sheep red blood cells as described previously (Kaplan & Clark, 1974 ). After T-cell depletion, the percentage of CD3+ cells was examined by using anti-CD3 antibody (Dako) by flow cytometry. The CD3+ cells accounted for less than 2% of the total lymphoid cells.
Monocyte depletion and CD34-positive selection from CBMNCs.
Monocytes were first depleted by incubating CBMNCs with silica (Immuno-Biological Laboratories) at 37 °C for 60 min under continuous rotation and by separating out phagocytic cells by Ficoll-Conray density gradient centrifugation. CD34+ haematopoietic progenitor cells were then isolated from the monocyte-depleted CBMNCs by using immunomagnetic Dynabeads M-450 (Dynal) covered with murine monoclonal antibodies (MAbs) against CD34 antigen. The Dynabeads were detached from the selected cells by incubation with antiserum against murine Fab fragments (DETACHaBEAD; Dynal) and the liberated cells were washed. Cells in each fraction (105106 cells) were resuspended in 50 µl flow buffer (PBS with 1% paraformaldehyde) and incubated with 10 µl anti-CD34phycoerythrin (PE) (Immunotech) for 30 min at 4 °C. After washing twice with PBS, the pellet was resuspended in 1 ml flow buffer. The percentage of CD34+ cells was analysed with a FACScan flow cytometer (Becton Dickinson) and CellQuest software.
Preparation of virus stocks.
CBMNCs were cultured for 3 days in the complete medium, i.e. RPMI-1640 medium supplemented with 10% foetal calf serum (FCS), 5 µg/ml phytohaemagglutinin (PHA; Difco) and 0·1 U/ml recombinant interleukin-2 (GIBCO BRL). The Z29 strain of HHV-6B, the U1102 strain of HHV-6A and the MRK strain of HHV-7 were used (Isomura et al., 1997 ). Activated fresh CBMNCs were mixed with CBMNCs infected with either HHV-6 or HHV-7 at a ratio of 1:5 and co-cultivated in the complete medium without PHA for 57 days. The supernatant of the infected culture containing a high virus titre was clarified by centrifugation at 3000 r.p.m. for 10 min and stored at -80 °C until use. For titration of virus stocks, the TCID50 of the virus stock was determined by infecting quadruplicate cultures of activated CBMNCs with serial 10-fold dilutions of virus stock as described previously (Yoshida et al., 1996
). Heat-inactivation was performed by incubating a virus sample at 56 °C for 1 h. Filter-depletion was done by passing a virus sample through an ultra-filtration system, Centricon 100 (Millipore), which eliminated materials with molecular masses of more than 100 kDa. No residual titre was detected after inactivation.
Infection of T-cell depleted CBMNCs.
T-cell depleted CBMNCs were resuspended in Iscoves modified Dulbeccos medium (IMDM) at a concentration of 1x106 cells/ml, inoculated with 5 ml of each virus sample at various m.o.i., centrifuged at 1500 g for 60 min to enhance adsorption and then washed twice with IMDM.
Colony assay for CFU-GM and BFU-E.
The infected and mock-infected cell pellets were finally resuspended in a semi-solid matrix (MethoCult GF H4434, StemCell Technology Inc., Vancouver, Canada) at a concentration of 5x104 cells/ml and plated in triplicate into a six-well tissue culture plate (Nunc) as described by Isomura et al. (1997) . The semi-solid matrix consisted of IMDM supplemented with 0·9% methylcellulose, 30% FCS, 1% BSA, 3 U/ml erythropoietin, 10-4 M mercaptoethanol, 2 mM L-glutamine, 50 ng/ml recombinant stem cell factor (SCF), 10 ng/ml recombinant granulocyte/macrophage-colony stimulation factor and 10 ng/ml recombinant interleukin-3. Plates were cultured in a 5% CO2 humidified atmosphere at 37 °C. Twelve days after plating, the numbers of CFU-GM and BFU-E colonies were counted.
TPO-inducible colony assay.
The infected and mock-infected cell pellets were finally resuspended in semi-solid matrix containing TPO at a concentration of 5x104 cells/ml and were plated in triplicate into a six-well tissue culture plate (Nunc). Two types of TPO-containing semi-solid matrix, serum-containing and serum-free matrices, were used. The serum-containing semi-solid matrix with TPO was prepared by adding 50 ng/ml TPO to a commercially available serum-containing semi-solid matrix (MethoCult H4230, StemCell Technology) consisting of IMDM supplemented with 0·9% methylcellulose, 30% FCS, 1% BSA, 10-4 M mercaptoethanol, 2 mM L-glutamine. The serum-free semi-solid matrix with TPO was prepared by adding 50 ng/ml TPO to a commercially available serum-free semi-solid matrix (MethoCult H4236, StemCell Technology), which includes IMDM supplemented with 0·9% methylcellulose, 10-4 M mercaptoethanol, 2 mM L-glutamine, 2% BSA, 200 µg/ml human transferrin and 10 µg/ml bovine pancreatic insulin. In some experiments, serum-free semi-solid matrix with TPO was further supplemented with 50 ng/ml SCF. The plates were cultured in a 5% CO2, 5% O2, 90% N2 humidified atmosphere at 37 °C for 10 days. Each colony was separately aspirated by a micro-capillary pipette and suspended in PBS. The cell suspension was cyto-spun at 650 r.p.m. for 5 min onto a poly-L-lysine (Miles-Yeda Ltd)-coated slide. The slide was fixed with 4% paraformaldehyde.
Identification of TPO-inducible colonies.
CFU-Meg colonies were identified on the basis of their translucent cytoplasm and highly refractile cell membrane (Muraoka et al., 1997 ; Yoshida et al., 1997
) under an inverted microscope. Colonies that did not fit the category were classified as non-CFU-Meg colonies. To confirm the identification made under the inverted microscope, we picked several colonies of each type and stained the constituent cells with Giemsa solution or immuno-stained them with a MAb (Dako) against the CD41 antigen, the human platelet/megakaryocyte-specific glycoprotein IIb/IIIa complex, by the alkaline phosphatase/anti-alkaline phosphatase technique (Muraoka et al., 1997
; Yoshida et al., 1997
).
In situ hybridization (ISH).
Digoxigenin-labelled HHV-6-specific probe was generated by using a 2·2 kbp BamHI fragment of the HHV-6 genome cloned in plasmid vector pUC18. Each specimen was covered with 10 µl hybridization solution containing 50% formamide, 10% dextran sulphate, 10 mM TrisHCl (pH 7·6), 1 mM EDTA, 600 mM NaCl, 1 mM EDTA, 1x Denhardts solution, 200 µg/ml E. coli tRNA and the probe and was placed on the bottom of a steel container floating in boiling water for 10 min. The slides were then incubated overnight at 42 °C in a moist chamber. The next day, these slides were washed at 50 °C, once with 2x SSC50% formamide, once with 2x SSC0·1% SDS and then twice with 0·1x SSC0·1% SDS. Detection of signal was performed by using a DIG nucleic acid detection kit (Boehringer Mannheim) according to the manufacturers recommendations.
Cytokines.
Recombinant human TPO and SCF were supplied by Kirin Brewery Co. Ltd (Tokyo, Japan).
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Results |
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Discussion |
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We encountered several technical difficulties during optimization of the culture conditions for CFU-Meg. Firstly, it is difficult to generate large macroscopic colonies. Many colonies of CFU-Meg never contain more than 50 cells, because megakaryocytes undergo several cycles of endoreduplication in which the DNA content is amplified but there is no cell division (Angchaisuksiri et al., 1996 ). In fact, we observed many hyperdiploid cells in the CFU-Meg colonies. Thus, in counting the colonies under an inverted microscope, we paid special attention not to overlook small colonies. Secondly, it is not possible to identify CFU-Meg colonies reliably on the basis of colony morphology alone. A small fraction (less than 10%) of the colonies identified as CFU-Meg were a 50:50 mixture of CD41-positive and CD41-negative cells, although the majority of the colonies identified as CFU-Meg were pure CFU-Meg colonies, as shown by the fact that more than 90% of the cells were CD41+. Several previous reports described the presence of mixed colonies of megakaryocytes and macrophages (Hogge et al., 1997
; Yoshida et al., 1997
).
Three culture conditions for TPO-inducible colony formation were selected according to previous reports (Hagiwara et al., 1998 ; Hogge et al., 1997
; Nishihira et al., 1996
). Human plasma was found to have an inhibitory effect on megakaryocyte colony formation and this effect was attributed to TGF-
in the plasma (Berthier et al., 1993
). Since then, serum-free conditions have been used routinely for megakaryocyte progenitor cells (Hagiwara et al., 1998
; Hogge et al., 1997
; Koizumi et al., 1998
; Schipper et al., 1998
; Tanimukai et al., 1997
; Yoshida et al., 1997
). However, Nishihira et al. (1993) reported the formation of large macroscopic CFU-Meg colonies under serum-containing conditions and Hagiwara et al. (1998)
explained that this was due to a synergistic effect of SCF in the serum with TPO. In our laboratory, large macroscopic colonies were readily generated in the serum-containing matrix and this condition was used to compare the effects of HHV-6A, HHV6B and HHV-7. A synergy of SCF and TPO (Kobayashi et al., 1996
) was suggested in our experiment under the serum-free condition with and without SCF. Furthermore, TPO induced both CFU-Meg and non-CFU-Meg colonies even under serum-free conditions without SCF or any other cytokines, indicating a multifunctional effect of TPO (Tanimukai et al., 1997
; Yoshida et al., 1997
).
We present evidence that HHV-6B, a more prevalent and clinically relevant variant of HHV-6 (Cone et al., 1999 ; Kadakia et al., 1996
; Wang et al., 1996
), suppresses all three lineages of haematopoietic colonies by demonstrating an m.o.i.-dependent reduction of BFU-E, CFU-GM and CFU-Meg colonies with the same source of CBMNCs. When comparing the growth of the three lineages, it is important to use the same lot of CBMNCs because of the wide variation in the number of haematopoietic progenitor cells in each lot. HHV-6A also had comparative suppressive effects on CFU-Meg colony formation at an m.o.i. of 1, although there are very few reports of the association of HHV-6A with graft failure after stem cell transplantation (Rosenfeld et al., 1995
). Recently, we monitored the activity of four herpesviruses (cytomegalovirus, HHV-6, HHV-7 and EpsteinBarr virus) after allogeneic peripheral blood stem cell transplantation and bone marrow transplantation and found that detection of the HHV-6 genome was associated with delayed engraftment, and was especially associated with the delayed recovery of platelets (Maeda et al., 1999
). Taken together, these data prompt us to recommend that constant vigilance of the activity of HHV-6 is needed after stem cell transplantation.
Two mechanisms have been presented to explain the suppression of bone marrow by herpesviruses: one is the direct effect of the viruses on haematopoietic progenitors (MacKintosh et al., 1993 ; Movassagh et al., 1996
; Rakusan et al., 1989
; Sindre et al., 1996
; Sing & Ruscetti, 1990
) and the other is an indirect effect through cytokine production by infected accessory cells (Apperley et al., 1989
; Busch et al., 1991
; Lagneaux et al., 1994
, 1996a
, b
; Mayer et al., 1997
; Steffens et al., 1998
). Thus, it is of particular interest to elucidate the interaction between HHV-6 and CD34+ cells, which are thought to be the major source of haematopoietic progenitor cells (Civin et al., 1984
; Leary et al., 1984
, 1985
). The detection of the HHV-6 genome in highly purified CD34+ cells by ISH, as well as the reduction of BFU-E, CFU-GM and CFU-Meg colonies by HHV-6 infection in semi-solid matrices without a feeder layer of accessory cells, support the former mechanism, i.e. HHV-6 has direct effects on haematopoietic progenitors. However, these data do not rule out the possibility that accessory cells or other types of bystander cells also have a role in the suppression of these cells. Recently, Luppi et al. (1999)
detected latent HHV-6 DNA by PCR in purified CD34+ cell fractions from leukopheresis products of HHV-6-seropositive donors. As the transplantation of purified CD34+ cells has been performed routinely (Cornetta et al., 1998
; Kawano et al., 1998
), it would be interesting to evaluate the comparative effects of herpesviruses including cytomegalovirus and HHV-6 on haematopoietic colony formation of the three lineages using the purified CD34+ cells.
In conclusion, we demonstrated quantitatively the suppressive effects of HHV-6 on colony formation of CFU-Meg. Using this system, we are planning to evaluate various antiviral agents (Yoshida et al., 1996 , 1998
) for the treatment of HHV-6-related myelosuppression.
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Acknowledgments |
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Received 19 May 1999;
accepted 3 November 1999.