Suppressive effects of human herpesvirus-6 on thrombopoietin-inducible megakaryocytic colony formation in vitro

Hiroki Isomura1, Mariko Yoshida1, Hikaru Namba1, Nobukiyo Fujiwara1, Reiko Ohuchi1, Fumio Uno1, Megumi Oda2, Yoshiki Seino2 and Masao Yamada1

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Two clinical observations, the association of human herpesvirus-6 (HHV-6) with delayed engraftment after stem cell transplantation and thrombocytopenia concomitant with exanthema subitum, prompted us to evaluate the suppressive effects of HHV-6 on thrombopoiesis in vitro. Different culture conditions for thrombopoietin (TPO)-inducible colonies in semi-solid matrices were examined. Using cord blood mononuclear cells as the source of haematopoietic progenitors, two types of colonies, megakaryocyte colony-forming units (CFU-Meg) and non-CFU-Meg colonies, were established. The former colonies were identified by the presence of cells with translucent cytoplasm and highly refractile cell membrane, most of which were positive for the CD41 antigen. Although the plating efficiency of both types was much higher under serum-containing conditions than under serum-free conditions, the proportion of CFU-Meg to non-CFU-Meg colonies was consistently higher under serum-free conditions. The plating efficiency of CFU-Meg colonies was doubled by adding stem cell factor to the serum-free matrix. The effects of two variants of HHV-6 (HHV-6A and 6B) and human herpesvirus-7 (HHV-7) on TPO-inducible colonies were then compared. HHV-6B inhibited both CFU-Meg and non-CFU-Meg colony formation under serum-free and serum-containing conditions. HHV-6A had similar inhibitory effects. In contrast, HHV-7 had no effect on TPO-inducible colony formation. Heat-inactivation and ultra-filtration of the virus sample completely abolished the suppressive effect. After infection of CD34+ cells with HHV-6, the viral genome was consistently detected by in situ hybridization. These data suggest that the direct effect of HHV-6 on haematopoietic progenitors is one of the major causes of the suppression of thrombopoiesis.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Human herpesvirus-6 (HHV-6) was first isolated from patients with lymphoproliferative disorders and AIDS (Salahuddin et al., 1986 ). Yamanishi et al. (1988) clearly demonstrated an aetiological relationship between primary HHV-6 infection and exanthema subitum, a self-limiting childhood disease characterized by fever and rash. The virus is predominantly T-lymphotropic (Lusso et al., 1991 ; Takahashi et al., 1989 ) and has been divided into two variants, HHV-6A and HHV-6B, on the basis of variations in restriction fragment profiles, growth properties and reactivity to monoclonal antibodies (Ablashi et al., 1991 ; Schirmer et al., 1991 ). Human herpesvirus-7 (HHV-7) is another T-lymphotropic herpesvirus. It was isolated by Frenkel et al. (1990) from the activated T cells of a healthy individual. As is the case with other herpesviruses, both HHV-6 and HHV-7 are thought to cause latent infection after primary infection in childhood and to reactivate either spontaneously or under immunosuppressive conditions. A variety of diseases, such as chronic fatigue syndrome, histiocytic lymphoadenopathy, mononucleosis-like syndrome and multiple sclerosis, have been reported to be associated with HHV-6 (Akashi et al., 1993 ; Buchwald et al., 1992 ; Soldan et al., 1997 ; Sumiyoshi et al., 1993 ).

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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Preparation of cord blood mononuclear cells (CBMNCs).
After obtaining consent from maternal patients, cord blood was collected and heparinized. The samples were diluted with Hanks’ balanced solution and were separated by Ficoll-Conray density gradient centrifugation. Low-density cells ({rho}<1·077 g/cm3) were collected and washed twice in PBS.

{blacksquare} 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.

{blacksquare} 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 (105–106 cells) were resuspended in 50 µl flow buffer (PBS with 1% paraformaldehyde) and incubated with 10 µl anti-CD34–phycoerythrin (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.

{blacksquare} 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 5–7 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.

{blacksquare} Infection of T-cell depleted CBMNCs.
T-cell depleted CBMNCs were resuspended in Iscove’s modified Dulbecco’s 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.

{blacksquare} 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.

{blacksquare} 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.

{blacksquare} 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 ).

{blacksquare} 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 Tris–HCl (pH 7·6), 1 mM EDTA, 600 mM NaCl, 1 mM EDTA, 1x Denhardt’s 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 SSC–50% formamide, once with 2x SSC–0·1% SDS and then twice with 0·1x SSC–0·1% SDS. Detection of signal was performed by using a DIG nucleic acid detection kit (Boehringer Mannheim) according to the manufacturer’s recommendations.

{blacksquare} Cytokines.
Recombinant human TPO and SCF were supplied by Kirin Brewery Co. Ltd (Tokyo, Japan).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Establishment of culture conditions for TPO-inducible colonies from megakaryocytic progenitors in CBMNCs
In a preliminary experiment using the serum-containing matrix, two types of TPO-inducible colonies, CFU-Meg and non-CFU-Meg colonies, were identified on the basis of their morphology under an inverted microscope (Fig. 1A). The former were tentatively identified by their translucent cytoplasm and highly refractile cell membrane (Muraoka et al., 1997 ; Yoshida et al., 1997 ), which are generally accepted as characteristics of megakaryoblasts. However, because this method was not completely reliable, colonies were separately picked and the constituent cells were characterized by immune histochemistry to confirm the identification (Fig. 1B). As a result, most of the cells obtained from ‘CFU-Meg’ colonies were positive for the CD41 antigen, while quite a few cells obtained from ‘non-CFU-Meg’ colonies were also positive for the same antigen. Overall concordance between the identification as ‘CFU-Meg’ colonies by morphology and CD41 positivity in the majority of constituent cells was not less than 90% in any of the experiments. Furthermore, Giemsa staining revealed that some of the cells obtained from CFU-Meg colonies had lobulated nuclei, which is an indicator of hyperploidy, which in turn is characteristic of megakaryocytic differentiation (Fig. 1C).



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Fig. 1. Morphology of two types of colonies, CFU-Meg and non-CFU-Meg colonies, formed in the presence of TPO. (A) Inverted microscopic view. (B) Immuno-staining with anti-CD41 antibody. (C) Giemsa staining of constituent cells. Magnification x20 (A), x132 (B) and x250 (C).

 
To optimize culture conditions for TPO-inducible colonies, we attempted to determine culture conditions under which larger and more CFU-Meg colonies and fewer non-CFU-Meg colonies would form. Three culture conditions, the serum-containing semi-solid matrix, the serum-free semi-solid matrix (without SCF) and the serum-free semi-solid matrix with SCF, were compared. Because plating efficiency varies considerably from lot to lot, in order to compare two conditions, we conducted a side-by-side comparison using the same lot of CBMNCs. As shown in Fig. 2(A), the plating efficiency of both CFU-Meg and non-CFU-Meg colonies was much higher in the serum-containing matrix than in the serum-free matrix. The number of CFU-Meg colonies with 50 or more cells, i.e. the number of colonies that were large enough to be examined macroscopically, was much greater in the serum-containing matrix. However, the proportion of CFU-Meg to non-CFU-Meg colonies was consistently higher in the serum-free matrix (4·2±2·5:1) than in the serum-containing matrix (1·35±0·28:1). The plating efficiency of CFU-Meg colonies was doubled by the addition of SCF to the serum-free matrix (Fig. 2B).



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Fig. 2. Numbers of CFU-Meg (filled bars, colonies consisting of 50 or more cells; open bars, colony consisting of fewer than 50 cells) and non-CFU-Meg colonies (shaded bars) after 10 days of incubation in semi-solid matrices containing 50 ng/ml TPO. (A) Numbers of colonies under serum-containing (+FCS) and serum-free (-FCS) conditions. (B) Numbers of colonies in the absence (-SCF) and presence (+SCF) of SCF under serum-free conditions. The results are presented as the means±SEM of triplicate cultures.

 
Effects of HHV-6A, HHV-6B and HHV-7 on TPO-inducible colony formation
The effects of two variants of HHV-6 (HHV-6A and HHV-6B) and HHV-7 on TPO-inducible megakaryocytic colony formation were then compared by using the serum-containing semi-solid matrix. T cell-depleted CBMNCs, which were used as the source of haematopoietic progenitor cells, were infected with one of these viruses and maintained in the semi-solid matrix. As shown in Fig. 3, HHV-6A and 6B inhibited TPO-inducible formation of both CFU-Meg and non-CFU-Meg colonies. In the cultures infected with HHV-6, however, some CFU-Meg and non-CFU-Meg colonies with sizes comparable to those observed in the mock-infected culture were clearly present. In contrast, HHV-7 had no effect on colony formation under these conditions.



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Fig. 3. Effects of HHV-6 and HHV-7 on TPO-inducible colonies. (A)–(E) Macroscopic images of CFU-Meg and non-CFU-Meg colonies in a semi-solid matrix 10 days after infection of T cell-depleted CBMNCs. (A) Mock-infected culture; (B)–(C) cultures infected with HHV-6B (Z29 strain) at an m.o.i. of 1 (B) or 10 (C) TCID50 per cell; (D) culture infected with HHV-6A (U1102) at an m.o.i. of 1; (E) culture infected with HHV-7 (MRK) at an m.o.i. of 1. (F) Number of CFU-Meg (filled bars, colonies consisting of 50 or more cells; open bars; colony consisting of fewer than 50 cells) and non-CFU-Meg (shaded bars) colonies after infection with HHV-6 and HHV-7. The results are presented as the means±SEM of triplicate cultures.

 
An m.o.i. dependency of the effect of HHV-6B was demonstrated both in the serum-containing and serum-free semi-solid matrices (Fig. 4A). To evaluate the suppressive effect of HHV-6B in the presence of other combinations of cytokines, similar experiments were performed in serum-free semi-solid matrix with or without SCF. Suppressive effects of HHV-6B on the CFU-Meg and non-CFU-Meg colonies were also found in the presence of SCF and TPO (Fig. 4B). Moreover, we confirmed the effect of HHV-6B on the CFU-GM and BFU-E colonies under comparable conditions by using two sets of the same sources of CBMNCs. HHV-6B suppressed all three lineages of haematopoietic colonies (Fig. 5). Heat-inactivation and ultra-filtration of the virus sample completely abolished the suppressive effect (Fig. 6), confirming that the suppression was due to the presence of infectious virus.



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Fig. 4. M.o.i.-dependent effects of HHV-6B (Z29) on CFU-Meg (filled bars, colonies consisting of 50 or more cells; open bars, colony consisting of fewer than 50 cells) and non-CFU-Meg colonies (shaded bars). (A) Numbers of colonies under serum-containing (+FCS) and serum-free (-FCS) conditions. (B) Numbers of colonies in the absence (-SCF) and presence (+SCF) of SCF under serum-free conditions. Cultures were either mock-infected (Mock) or infected at an m.o.i. of 1 (MOI 1) or 10 (MOI 10) TCID50 per cell. The results are presented as the means±SEM of triplicate cultures.

 


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Fig. 5. Numbers of CFU-GM, BFU-E and CFU-Meg colonies in semi-solid culture after infection of two sets of the same sources of CBMNCs (A, lot #14; B, lot #15) with HHV-6B (Z29) at an m.o.i. of 10 TCID50 per cell. The results are presented as the means±SEM of triplicate cultures.

 


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Fig. 6. Numbers of CFU-Meg (filled bars, colonies consisting of 50 or more cells; open bars, colony consisting of fewer than 50 cells) and non-CFU-Meg colonies (shaded bars) after infection with intact or heat-inactivated HHV-6 at an m.o.i. of 10 TCID50 per cell equivalent (A) and with intact or filter-depleted HHV-6 at an m.o.i. of 10 TCID50 per cell equivalent (B). The results are presented as the means±SEM of triplicate cultures.

 
Detection of the HHV-6 genome in CD34+ cells
In order to elucidate the interaction between HHV-6 and haematopoietic progenitors in CBMNCs, we purified CD34+ cells and infected them with HHV-6B. Selection of CD34+ cells from monocyte-depleted CBMNCs was performed by using magnetic beads (Fig. 7A). The purified cells were infected with the Z29 strain of HHV-6B at an m.o.i. of 10 TCID50 per cell. These cells were maintained in IMDM supplemented with 30% FCS for 72 h. We next re-selected CD34+ cells by using magnetic beads. At this point, we obtained more than 99% purity. We detected the HHV-6 viral genome in pure CD34+ cells by ISH (Fig. 7B).



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Fig. 7. Detection of the HHV-6 genome by ISH in CD34+ cells. (A)–(C) Flow-cytometry of CD34 antigen expressed on cells selected with magnetic beads from CBMNCs (A), on cells infected with HHV-6 and maintained for 72 h after first selection (B) and on re-selected cells after 72 h incubation (C). (D) Detection of the HHV-6 genome by ISH in re-selected CD34+ cells. The arrows indicate hybridization signals, while the arrowheads indicate magnetic beads used for the selection. Magnification x200.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
In this paper, we demonstrate for the first time the suppressive effect of HHV-6 on megakaryocyte progenitor cells by using an in vitro CFU-Meg colony assay. Colony assays in a semi-solid matrix have proven to be beneficial in the quantification of haematopoietic progenitor cells of erythroid and granulocyte/macrophage lineages (Broxmeyer et al., 1989 ; Fauser & Messner, 1978 ; Nakahata & Ogawa, 1982 ). However, the culture conditions for megakaryocyte progenitors were very hard to establish until the discovery of the genuine megakaryocyte colony-stimulating factor, TPO (Bartley et al., 1994 ; de Sauvage et al., 1994 ). We owe our recent advance in the CFU-Meg colony assay to the cloning of the TPO gene and mass-production of recombinant TPO (Kaushansky et al., 1994 ; Wendling et al., 1994 ). The CFU-Meg colony assay was first applied to evaluate the suppressive effect of human immunodeficiency virus on megakaryopoiesis (Zauli et al., 1991 ) and quantitative in vitro data, which is well in accord with clinical observations, has been obtained (Cole et al., 1998 ; Zauli et al., 1992 ).

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-{beta} 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 Epstein–Barr 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.


   Acknowledgments
 
We thank Kirin Brewery Co. Ltd for technical advice on the CFU-Meg colony assays and Dr K. Tabuchi for helpful discussions and encouragement. Part of this research project was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture, Japan.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 19 May 1999; accepted 3 November 1999.