Institute of Virology and Immunoprophylaxis, Sensemattstrasse 293, CH-3147 Mittelhäusern, Switzerland1
Laboratory of Bioengineering, National Institute of Animal Health, Ibaraki 305, Japan2
Author for correspondence: Artur Summerfield. Fax +41 31 8489222. e-mail artur.summerfield{at}ivi.admin.ch
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Abstract |
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Introduction |
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Methods |
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Cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) foetal calf serum (FCS, Sigma), 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 39 °C. Recombinant porcine granulocytemacrophage colony-stimulating factor (GM-CSF, 5 ng/ml; Inumaru & Takamatsu, 1995 ) or lipopolysaccharide (LPS, 1 µg/ml; Sigma) was added to stimulate BMHC proliferation.
Bone marrow stroma cells (BMSC) were generated by culture of adherent BMHC for 23 weeks in DMEM supplemented as described above. Non-adherent BMHC were removed after 3 and 7 days of incubation, in combination with a medium change. After a further 12 weeks, a monolayer of cells had developed with mainly fibroblast, endothelial and macrophage morphology.
Bone marrow-derived macrophages (BM-M) were generated by culture of BMHC in DMEM supplemented with 20% (v/v) FCS and 20% (v/v) heparinized porcine plasma (all other components were as mentioned above) for 10 days at 39 °C. Non-adherent BMHC were removed after 3 and 7 days of incubation in combination with a medium change.
Virus infection.
The moderately virulent CSFV strain Alfort/187 (kindly provided by K. Depner, Tierärztliche Hochschule Hannover, Germany; Summerfield et al., 1998b ) and the virulent strain Brescia (kindly provided by H.-J. Thiel, University of Giessen, Germany; Summerfield et al., 1998a
) were propagated in mycoplasma-free SK-6 cells as described previously (Knoetig et al., 1999
). Cell lysates were prepared by sonication and were then clarified by centrifugation. Mock-treated controls were prepared in the same manner from non-infected SK-6 cells. UV-inactivated controls were prepared by exposing the cell lysate preparations to a 40 W UV lamp at a distance of 10 cm for 20 min (virus inactivation was controlled). BMHC were infected with CSFV, mock or UV-inactivated preparations on ice at an m.o.i. of 1 TCID50 per cell unless otherwise specified. The inoculum was removed after 1 h by centrifugation and the cells were washed twice with PBS-A (350 g, 10 min, 4 °C). Virus titres were determined and calculated as described previously (Knoetig et al., 1999
).
Monoclonal antibodies (MAbs) and flow cytometry (FCM).
For definition of BMHC populations, anti-SWC3 (MAb 74-22-15, IgG1; Saalmüller, 1996 ) and anti-SWC8 (MAb MIL3, IgM, Serotec; Haverson et al., 1994
; Saalmüller, 1996
) MAbs were used. By an SWC3/SWC8 double immunofluorescence analysis of BMHC, the granulocytic lineage was identified as SWC3+ SWC8+, the monocytic cells as SWC3+ SWC8- and the early myeloid precursors as SWC3low SWC8- (Summerfield & McCullough, 1997
). Fas expression was determined by using anti-Fas MAb CH-11 (Upstate Biotechnology). Indirect MAb labelling for triple immunofluorescence FCM was performed in a three-step procedure with isotype-specific conjugates [goat anti-mouse IgG, F(ab')2 fragments, FITC- or PE-conjugated or biotinylated; Southern Biotechnology Associates] and streptavidinSpectralRed conjugate (Southern Biotechnology Associates) as described previously (Summerfield & McCullough, 1997
).
Interferon- (IFN-
) was neutralized with a pool of MAbs (K9 and F17, each 1 µg/ml; Diaz de Arce et al., 1992
). B. Charley and C. La Bonnardière (INRA, France) kindly contributed the interferon reagents. A MAb against GM-CSF (64-10) was generated in our laboratory by established methods (Butcher et al., 1991
). Hybridoma supernatant from MAb 64-10 was used at 25% (v/v), a concentration found to block GM-CSF-induced BMHC proliferation (A. Summerfield, H. Gerber and K. C. McCullough, unpublished data).
Cell sorting.
For immunomagnetic cell separation, BMHC were labelled using the following incubation steps: (i) MAb; (ii) biotinylated goat anti-mouse Ig (Jackson ImmunoResearch Laboratories); and (iii) biotinylated paramagnetic microbeads (Miltenyi Biotec). Subsequently, the cells were sorted using the MACS magnetic cell separation system, following the manufacturers instructions (Miltenyi Biotec). After cell separation, the purity of the positive and negative fractions was controlled by FCM analysis (found to be over 95%).
CSFV detection.
For detection of CSFV-infected cells, MAbs against the CSFV structural glycoprotein E2 (MAb HC/TC26, 10 µg/ml; kindly provided by Dr Bommeli AG, Switzerland; Greiser-Wilke et al., 1990 ) and the CSFV non-structural protein p125 (MAb C16, kindly provided by I. Greiser-Wilke, Hannover Veterinary School, Germany) were used. For this procedure, the cells were fixed and permeabilized (Cell permeabilization kit, Harlan Sera-Lab) before labelling with the MAb. This method, combined with the m.o.i. employed, permitted the detection of de novo E2 synthesis only, and not the input virus (Knoetig et al., 1999
).
CSFV RNA was detected by RTPCR after extraction of RNA with Trizol (Gibco BRL). The RTPCR employed the Titan RTPCR system (Boehringer Mannheim), according to the manufacturers instructions, with 1 µg RNA template plus 0·4 µM sense and anti-sense primers for CSFV (HCV-1, 5' CCG TGA CCG TGG TAG GGG AAA 3', and HCV-2, 5' ATT TGG TCT TCG AGG CGC AGCA 3'; Wirz et al., 1993 ) and porcine
-actin (5' GGA CTT CGA GCA GGA GAT GG 3' and 5' GCA CCG TGT TGG CGT AGA GG 3'). The latter were internal controls for the amount of input RNA. Reverse transcription was performed at 50 °C for 30 min followed by 25 cycles of PCR for DNA amplification (94 °C for 45 s; 25 cycles of 30 s at 94 °C, 30 s at 59 °C and 1 min at 68 °C).
Cell viability and apoptosis analysis.
For quantification of apoptotic cells expressing phosphatidylserine (PS) on their surface and dead cells permeable to propidium iodide (PI, Sigma), dual-parameter analysis of annexin VFITC (Bender Med Systems) and PI were performed (Vermes et al., 1995 ). To this end, 5x105 cells were labelled with 2 µg/ml annexin VFITC in 140 mM NaCl, 2·5 mM CaCl2, 10 mM HEPES (pH 7·4) for 10 min. After FL1/FL2 compensation, PI (100 ng/ml) was added, in order to discriminate between apoptotic and dead cells, and the sample was acquired by FCM.
A second analysis of apoptosis used a method based on cellular DNA loss, typically found in late-stage apoptosis. Cells were fixed with 75% (v/v) ethanol (0 °C, 2 min) followed by washing and centrifugation. DNA was stained with 50 µg/ml PI plus 100 µg/ml RNase for 30 min at 37 °C and cells were then analysed by FCM. The DNA histograms obtained were used to quantify the apoptotic cells located in the sub-G1 region (Darzynkiewicz et al., 1992 ).
The mitochondrial transmembrane potential (m) of BMHC was measured by incubation with 40 nM 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] (Molecular Probes) for 10 min at 37 °C (Zamzami et al., 1995
). As negative controls, cells were treated with the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (50 µM; Sigma).
Formation of reactive oxygen species (ROS) was quantified by incubation of BMHC with hydroethidine (HE, 5 nM; Molecular Probes) for 10 min at 37 °C (Rothe & Valet, 1990 ). During the reaction, HE is oxidized to ethidine, which emits a red fluorescence.
Nuclear morphology was determined by fluorescence microscopy of cytospin preparations (200 g, 5 min) of BMHC, which were stained with the DNA dye Sytox green (Molecular Probes).
Caspase activity.
Caspase activity from cell lysates was tested with fluorogenic substrates composed of peptides conjugated to 7-amino-4-(trifluoromethyl)coumarin (AFC) or 7-amino-4-methylcoumarin (AMC) as described previously (Summerfield et al., 2000 ). Substrates were Z-DEVD-AMC for caspase 3, Ac-IETD-AFC for caspase 8 and Ac-LEHD-AFC for caspase 9 (all from Calbiochem). Briefly, 50 µg protein obtained from cell lysates was mixed with 5 mM of the enzyme substrate in assay buffer [312·5 mM HEPESNaOH, 31·25% (w/v) sucrose, 10 mM DTT, 0·3125% (w/v) CHAPS, pH 7·5] to obtain a final volume of 200 µl. As negative controls, samples were incubated for 1 h at 37 °C with specific caspase inhibitors (Z-VAD-FMK as general caspase inhibitor, Z-DEVD-FMK as caspase 3 inhibitor, Z-IETD as caspase 8 inhibitor and Z-LEHD-FMK as caspase 9 inhibitor), before addition of the corresponding substrate. After incubation for 2 h at 30 °C, fluorescence was read in a Spectramax Gemini (Molecular Devices). Enzyme activity was calculated by subtraction of the amount of AFC/AMC released in the negative controls from that obtained with the test samples and expressed as pmol AMF/AMC released per min per µg protein.
ROS scavengers.
The potential role of ROS in the effect of CSFV on the viability of BMHC was investigated by using the ROS scavengers (all from Sigma) catalase (CAT, 1000 U/ml), superoxide dismutase (SOD, 2200 U/ml), butylated hydroxyamisol (BHA, 100 µM) and N-acetylcysteine (NAC, 2 mM). CAT, BHA and NAC have been demonstrated to affect extracellular and intracellular ROS activity (Sundaresan et al., 1995 ; Katschinski et al., 2000
; Schweizer & Peterhans, 1999
; Zaragoza et al., 2000
; Shimura et al., 2000
).
PKH labelling for co-culture experiments.
BMHC were labelled by using the red fluorescence cell linker kit PKH-26 GL (Sigma), following the manufacturers instructions. Labelled BMHC were detected in the fluorescence-3 channel of the FCM and thereby could be excluded when co-culture experiments with unlabelled cells were analysed.
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Results |
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This infection of BMHC cultures resulted in high titres of extracellular progeny virus. Titres of 107108 TCID50/ml were measured at 3 days p.i. after infection with both the Alfort/187 and Brescia strains.
Induction of apoptosis in CSFV-infected BMHC cultures
Analysis of BMHC obtained from CSFV-infected pigs revealed an increase in both necrotic cells and cells undergoing apoptosis (Summerfield et al., 2000 ). Consequently, the influence of CSFV on the survival of BMHC in vitro was investigated. After infection of BMHC at an m.o.i. of 1 TCID50 per cell, increases in the numbers of both dead and definable apoptotic cells were found at 48 h p.i., compared with mock-treated cultures (Fig. 2a
). In fact, the elevated levels of apoptosis in CSFV-infected cultures were seen between days 2 and 4 of the culture, often with a maximum at 3 days p.i., when reduced cell counts (3065%) were also found. The number of annexin V+ PI- apoptotic cells following infection was four times that of control mock-treated cultures (lower-right quadrants) and the number of dead, PI+ cells was also higher in infected BMHC (upper-right quadrants). These results were confirmed by the observation that CSFV-infected BMHC cultures displayed an increased number of cells with reduced
m compared with mock-treated controls (Fig. 2b
; left quadrants). In addition, over half of these definable apoptotic cells displayed increased ROS activity, as demonstrated by oxidation of HE to ethidine (Fig. 2b
; upper quadrants). As further confirmation of a possible role for apoptosis during CSFV infection of BMHC, the DNA content of the above cells was analysed. Within the infected cultures, more cells were located in the sub-G1 region (Fig. 2c
).
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No significant differences were found in the number of cells with reduced viability at the different m.o.i. employed, 0·1, 1 and 10 TCID50 per cell (data not shown). Furthermore, no significant differences were observed when the degrees of virus-induced apoptosis with the moderately virulent Alfort/187 strain and the highly virulent Brescia strain were compared (data not shown).
Influence of BMHC stimulation on culture characteristics following CSFV infection
BMHC cultures require certain haematopoietic growth factors for their survival, such as GM-CSF (Williams et al., 1990 ). Cultures without additional stimulation suffered from relatively high rates of spontaneous apoptosis (1030% annexin V+ cells in the mock-treated cultures, Table 1
). Addition of either GM-CSF or LPS improved the viability of both mock-treated and virus-infected cultures (Table 1
). However, the ratio of annexin V+ cells in infected BMHC increased compared with mock-treated BMHC. This related to an increase in the percentage of cells infected (Table 1
). When GM-CSF bioactivity was blocked by an anti-GM-CSF neutralizing antibody, the level of apoptosis increased in both mock- and virus-infected cultures to that seen in the absence of stimuli, as did the number of virus-infected cells. It was also noted that the presence of both LPS and anti-GM-CSF antibodies reversed the influence of LPS, demonstrating the role of GM-CSF in the LPS-induced effect.
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As shown in Table 2, when the BMHC were stained with annexin V after 24 h, the difference between the cultures treated with supernatant from mock- and virus-infected BMHC was not significant. There was a slight increase of annexin V+ BMHC cells when the supernatants were from CSFV-infected BMHC, and this increase was neutralized by addition of anti-E2 MAb. When the supernatants were from mock- or virus-infected BM-M
or BMSC, there was no difference in the number of annexin V+ cells in the treated BMHC at 24 h.
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The role of ROS, which are also potential soluble mediators of apoptosis, was investigated by using different ROS scavengers. These included CAT, SOD and BHA. None of these scavengers reduced the observed virus-induced cell death, as exemplified in Table 2 by the data obtained with BHA.
An additional experiment confirmed that soluble factors were not the elements mainly responsible for the observed CSFV-induced cell death in BMHC. In place of supernatant transfer, the infected BMHC were co-cultured with fresh indicator BMHC, but were separated from the latter by using a trans-well culture system. No difference was seen between co-cultures with mock-treated and virus-infected BMHC (Table 2). In fact, the slight increase in apoptosis observed when the supernatants from virus-infected BMHC were employed was no longer evident.
Role of cognate interactions
Taken together, the above results demonstrated that CSFV-induced BMHC death was not a direct consequence of the presence of CSFV, but also was not induced by soluble factors released by infected cells. Consequently, the possible role of cognate interactions between CSFV-infected and non-infected BMHC in virus infection-induced apoptosis was analysed. To this end, BM-M and fibroblastoid BMSC were employed. These two types of cell are known to be amongst the main regulatory elements in the bone marrow microenvironment (Dorshkind, 1990
). After 48 h of culture, the cells were harvested and co-cultured with freshly isolated BMHC. In order to inactivate the virus and inhibit its intercellular spread, infected cells in particular co-cultures were fixed with 1% (w/v) paraformaldehyde. The viability of the indicator BMHC at 24 h after co-culture revealed no significant differences between co-cultures with mock-treated or with CSFV-infected BM-M
and BMSC (Table 3
). In contrast, by 72 h after co-culture, apoptosis was clearly enhanced when CSFV-infected cells were used. This was less evident when the BM-M
and BMSC were fixed before co-culture.
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Discussion |
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Concerning the influence of CSFV infection on BMHC, several features of apoptotic cell death were identified: PS expression on the cell surface, reduced m, increased ROS generation, loss of DNA and increased caspase activity. The particularly strong increase in caspase 9 in CSFV-infected cultures would point to a role for the mitochondrion-initiated pathway of apoptosis (Budihardjo et al., 1999
; Rathmell & Thompson, 1999
). It is clear that CSFV-induced BMHC death does not relate to the apoptosis observed with another pestivirus, the cytopathic form of bovine viral diarrhoea virus (Zhang et al., 1996
; Hoff & Donis, 1997
; Schweizer & Peterhans, 1999
; Jungi et al., 1999
). This may be expected, considering that CSFV strains, such as those employed in the present study, are primarily non-cytopathogenic for kidney cell lines, M
and fibroblastoid BMSC. Although Shimizu et al. (1995)
reported a cytopathic effect of CSFV in cultured BMSC, we have never observed this phenomenon. Of course, the effect on the stromal cells in vitro reported by Shimizu et al. (1995)
was only detectable late (10 days) p.i., a time-point well beyond our observations on the induction of apoptosis.
The majority of apoptotic BMHC in CSFV-infected cultures were uninfected, indicating that this effect of CSFV on BMHC was not a direct consequence of virus infection and replication. A possible explanation would be that infected cells were modulated in their haematoregulatory activity. Important therein are the cellular components of the bone marrow stroma. However, no role could be identified for factors or cognate interaction between BMHC and infected M or other stromal cells, at least in the context of induction of apoptosis in the BMHC. Furthermore, none of our investigations pointed to a role for soluble factors released from infected cells in CSFV-induced apoptosis of BMHC.
Interestingly, an increased rate of apoptosis was observed when uninfected BMHC were co-cultured with virus-infected BMHC. This effect was dependent on cell contact between infected and uninfected cells. Such an observation would implicate death receptors on BMHC interacting with their ligands on CSFV-infected BMHC, but not stromal cells.
Taken together, the present results describe an in vitro model for the investigation of the characteristic bone marrow atrophy and peripheral loss of mature granulocytes found in CSF. The observed CSFV-dependent induction of apoptosis in granulocytic cells appears to relate to observations made with BMHC isolated from CSFV-infected pigs (Summerfield et al., 2000 ). This cell death could be central to the observed loss of mature granulocytes in the blood during CSF, being replaced by immature granulocytes, and in the development of bone marrow atrophy (Summerfield et al., 2000
). The experimental system employed will prove particularly useful for further analyses of CSFV pathogenesis and for the study of CSFV gene products involved in leukocyte modulation and apoptosis.
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Acknowledgments |
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Footnotes |
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References |
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Received 2 November 2000;
accepted 23 February 2001.