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Journal of General Virology (2000), 81, 881–887.

Printed in Great Britain


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Bovine viral diarrhoea virus and bovine herpesvirus-1 prime


uninfected macrophages for lipopolysaccharide-triggered
apoptosis by interferon-dependent and -independent
pathways
L. Perler, M. Schweizer, T. W. Jungi and E. Peterhans
Institute of Veterinary Virology, University of Berne, Laenggass-Str. 122, CH-3012 Berne, Switzerland

The flavivirus bovine viral diarrhoea (BVD) virus exists in two biotypes, cytopathic (cp) and non-
cytopathic (ncp), defined by their effect on cultured cells. Cp BVD virus-infected cells undergo
apoptosis and may promote apoptosis in uninfected cells by an indirect mechanism. Macrophages
(Mφ) infected with cp, but not ncp, BVD virus release a factor(s) in the supernatant capable of
priming uninfected Mφ for activation-induced apoptosis in response to lipopolysaccharide. A
possible role of interferon (IFN) type I was suggested previously by the observation that this
cytokine primed for activation-induced apoptosis and was present in supernatants of Mφ infected
with cp, but not ncp, BVD virus. Here, supernatants of both Mφ infected with a wider range of cp
BVD virus and Mφ infected with bovine herpesvirus-1 are shown to contain such priming activity.
Two lines of evidence indicate that factors in addition to IFN type I prime uninfected Mφ for
apoptosis. First, supernatants of Mφ infected with cp BVD virus contained much less IFN than is
required for priming for apoptosis. Second, whereas antiviral activity was neutralized by a vaccinia
virus-encoded IFN type I receptor, B18R, the capacity of the supernatant to prime for apoptosis
was unaffected by this treatment. The apparent molecular mass of the factor(s) priming for
apoptosis was between 30 and 100 kDa. Priming of uninfected cells for activation-induced
apoptosis may add a new facet to virus pathogenesis and may contribute to the formation of lesions
not related directly to virus replication.

Introduction This virus is of particular interest in studies of virus-induced


Viruses have evolved strategies to induce or prevent apoptosis because of the existence of closely related ‘ pairs ’ of
apoptosis in their host cells. Generally, virus-induced apoptosis cytopathic (cp) and non-cytopathic (ncp) biotypes (Weiss et al.,
is believed to enhance the release of virus progeny from 1994 ; Paton, 1995). The two biotypes of such a pair differ only
infected cells. It may, however, also represent a host defence in one non-structural protein, NS23, which is cleaved into NS2
mechanism, as apoptotic cells cease to produce virus progeny. and NS3 in cp, but not ncp, biotypes (Brownlie, 1990 ; Meyers
Virus inhibition of apoptosis may therefore contribute to & Thiel, 1996). Moreover, both biotypes of BVD virus are
prolonged infection (for reviews see Knipe, 1996 ; Evan & associated with mucosal disease, a lethal form of infection with
Littlewood, 1998 ; O’Brien, 1998 ; Tschopp et al., 1998). BVD virus. It is observed in animals infected persistently with
Bovine viral diarrhoea (BVD) virus is a pestivirus of the ncp BVD virus from the early period of their intrauterine
family Flaviviridae (Baker, 1987 ; Nettleton & Entrican, 1995). development. The cp biotype of BVD virus detected in animals
with mucosal disease may be the result of a mutation of the ncp
Author for correspondence : Ernst Peterhans. biotype (hence the term ‘ virus pair ’), or may be due to
Fax j41 31 631 2534. e-mail ernst.peterhans!ivv.unibe.ch superinfection. The cp biotype is believed to kill these
immunotolerant animals because their immune systems fail to
The GenBank accession numbers of the BVD virus sequences reported control virus multiplication (Brownlie, 1990 ; Meyers & Thiel,
in this paper are AF117699–AF117706. 1996). Cells infected with cp BVD virus have been shown to

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L. Perler and others

undergo apoptosis (Zhang et al., 1996), which is associated virus infectivity was purchased from Sigma. RboIFN-α was generously
with cleavage of poly(ADP-ribose) polymerase (Hoff & Donis, provided by Novartis.
1997 ; Schweizer & Peterhans, 1999) and can be prevented
Cells. Mφ were obtained from the blood of Red Holstein cows as
by selected antioxidants (Schweizer & Peterhans, 1999). In described previously (Jungi et al., 1997). Briefly, peripheral bovine
addition to inducing apoptosis in its host cells, BVD virus may mononuclear cells were isolated by using Ficoll–Hypaque centrifugation
also influence the viability of uninfected cells, via factors and Mφ were permitted to differentiate by culturing the cells for 7 days
under non-adherent conditions in Teflon bags at a cell concentration of
released from infected cells. An example of such factors are
4i10' cells\ml in Iscove’s modified Dulbecco’s minimum essential
interferons (IFNs), which have been shown to have anti- medium (MEM) supplemented with HEPES (10 mM), nonessential amino
as well as pro-apoptotic properties (Rodriguez-Villanueva & acids for MEM (Life Technologies, 1 %), vitamins for MEM (Life
McDonnell, 1995 ; Yanase et al., 1998). Specifically, IFNs have Technologies, 1 %), streptomycin (100 µg\ml), penicillin (100 IU\ml),
been shown to prime for apoptosis via increased synthesis of amphotericin B (2n5 µg\ml), -glutamine (2 mM), 2-mercaptoethanol
2h,5h-oligoadenylate (2-5A) and its activation of RNase L (Der (50 µM) and 20 % FCS. Madin Darby bovine kidney (MDBK) cells and
et al., 1997 ; Diaz-Guerra et al., 1997 ; Castelli et al., 1998) or via Sf9 insect cells were obtained from the American type culture collection
inhibition of protein synthesis by PKR (Jagus et al., 1999). (Manassas, VA, USA). MDBK cells of passages 120 to 140 were used for
IFN type I assay. BVD and BHV-1 viruses were grown and titrated on
Apoptosis of uninfected NIH3T3 cells was observed when bovine embryonic turbinate cells. These cells were prepared from
RNase L was overexpressed by means of an inducible promoter foetuses obtained from a local abattoir and were maintained in MEM
or activated by 2-5A or double-stranded RNA (Castelli et al., supplemented with 2 % FCS, -glutamine (2 mM), penicillin (100 IU\ml)
1998). In contrast, inhibition of the 2h,5h-oligoadenylate and streptomycin (100 µg\ml) at 37 mC in a humidified 5 % CO
#
synthetase\RNase L system prevented apoptosis in response atmosphere. All batches of cells used in our experiments were found to
to double-stranded RNA (Rivas et al., 1998). Pre-treatment of be free of BVD virus by immunofluorescence testing.
mice with IFN-γ was shown to enhance lipopolysaccharide
Viruses. BHV-1 (Colorado strain) was originally obtained from V.
(LPS)-triggered apoptosis markedly in thymocytes, whereas Bitsch (Aarhus, Denmark). The recombinant baculovirus vector ex-
the simultaneous injection of anti-IFN-γ antibody and LPS pressing B18R and the vector control were kindly provided by G. L.
prevented apoptosis (Kato et al., 1997). In vitro, human Smith (Sir William Dunn School of Pathology, Oxford, UK) and have
been described elsewhere (Alcamı! & Smith, 1992 ; Symons et al., 1995).
immunodeficiency virus type 1 (HIV-1)-infected macrophages
The BVD virus strains used included NADL, TGAC (Ridpath et al., 1991)
(Mφ) enhanced dramatically the proportion of cells undergoing and SuwaCP (all cp) and TGAN (Ridpath et al., 1991) and SuwaNCP
apoptosis of uninfected but not infected T cells (Herbein et al., (both ncp). The TGAC and TGAN virus strains were kindly provided by
1998). These authors postulate an indirect mechanism of V. Moennig (Hannover, Germany), whereas SuwaCP and SuwaNCP are
apoptosis in uninfected T cells mediated by antigen-presenting a ‘ virus pair ’ isolated from peripheral blood of an animal suffering from
cells. Furthermore, in porcine reproductive and respiratory mucosal disease. Separation and biological cloning of the two Suwa BVD
syndrome virus infection, it is mainly uninfected bystander virus biotypes was performed by the plaque formation method using a
cells that undergo apoptosis, rather than infected cells. In vivo methyl cellulose overlay medium consisting of 1n5 % methyl cellulose in
MEM supplemented with penicillin (100 IU\ml) and 2 % FCS (Nakamura
studies showed the apoptotic cells to consist predominantly of et al., 1993).
mononuclear cells (Sirinarumitr et al., 1998). The genetic relationship of SuwaCP and SuwaNCP BVD viruses was
Supernatants of Mφ infected with cp BVD virus that prime determined by sequencing using standard techniques and an ABI
uninfected Mφ for apoptosis were found to contain IFN (Adler PRISM 310 Genetic analyser (Perkin Elmer). The following primers were
et al., 1997). Moreover, recombinant bovine (rbo) IFN-αI.1 used for amplification of BVD virus cDNA : 5h-UTR, 5h GAGGCTA-
primes for apoptosis (Adler et al., 1995). Therefore, we GCCATGCCCTTAG 3h (sense) and 5h TCAACTCCATGTGCCAT-
investigated whether it is the IFNs that are responsible for GTAC 3h (antisense) ; E2, 5h GACAGGGACTGTGAGCTGTA 3h (sense)
and 5h GGCCCCTCACTTGATATGAT 3h (antisense) ; and NS23, 5h
priming by supernatants of cp BVD virus-infected Mφ. We GAAGTCTACGGCATGCCAAA 3h (sense) and 5h ACTGGGGCT-
show that priming was due to a factor(s) distinct from, or in CTGGGTGTGGT 3h (antisense). Analysis of the sequences revealed
addition to, IFNs. The presence of such a factor(s) in that, with the exception of four point mutations in the NS23 region, the
supernatants of Mφ infected with different strains of cp BVD nucleotide sequences of the two virus genomes were identical. One of
virus and bovine herpesvirus-1 (BHV-1), a cp DNA virus, these mutations was silent, whereas the other three led to changes in the
suggests that priming for activation-induced apoptosis may be amino acid sequence. Northern blotting of genomic viral RNA performed
of more general significance in virus–host interactions. by hybridization with virus strain-specific DIG-labelled RNA probes
revealed that the genomes of SuwaCP and SuwaNCP BVD viruses were
both approximately 12n5 kb in size (results not shown).

Induction of apoptosis. Mφ seeded in 25 cm# culture flasks at a
Methods density of 5i10' cells per flask were infected with the appropriate strain

Reagents. Foetal calf serum (FCS) and cell culture media for bovine of BVD virus or BHV-1 in 1n5 ml culture medium without FCS at an m.o.i.
cells were obtained from Seromed (Biochrom) and culture media for of 1 for 1 h at 37 mC. After adsorption of the virus, the inoculum was
insect cells were bought from Life Technologies. FCS was free of BVD removed by washing the cells in culture medium without FCS prior to the
virus and antibody to BVD virus, as tested by virus isolation and serum addition of complete medium with 2 % FCS. Supernatants were harvested
neutralization assay, respectively. β-Propiolactone for inactivation of after 48 h incubation at 37 mC and virus was inactivated by incubation

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Virus-induced factors prime for apoptosis

with β-propiolactone at a 4000-fold dilution for 18 h at 4 mC followed by


2 h at 37 mC (Barrett et al., 1984 ; Perler et al., 1999). Virus inactivation
was verified by titration on bovine turbinate cells. Mφ seeded in 24 well
plates at a density of 5i10& cells per well were incubated with the
inactivated supernatant (400 µl per well) and fresh culture medium with
2 % FCS (400 µl per well) for 48 h at 37 mC. The inoculum was replaced
by fresh culture medium containing 1 µg\ml LPS. Apoptosis was
measured after 48 h incubation.

IFN assay. Procedures for measuring biological activity of type I


IFN in supernatants of virus-infected bovine monocyte-derived Mφ were
described previously. The assay used is based on the reduction of Sendai
virus growth by IFN as determined by immunocytochemistry (Perler et Fig. 1. IFN type I activity is restricted to supernatants of cp BVD virus-
al., 1999). infected bovine monocyte-derived Mφ. Monocyte-derived Mφ were mock-
infected or infected with different strains of cp (NADL, TGAC, SuwaCP)

Apoptosis assay. The fragmentation of cellular DNA was analysed and ncp (TGAN, SuwaNCP) BVD virus or BHV-1. Biological activity of IFN
quantitatively by FACS analysis according to Cossarizza et al. (1995). type I contained in undiluted (BVD virus) or 1 : 1000-diluted (BHV-1)
Detached cells were collected by centrifugation and the pellet was lysed supernatants of the infected macrophages was measured after 48 h. IFN
with 500 µl lysis buffer (0n1 M sodium citrate, pH 6n5, 1 % Triton X-100 type I is expressed as the concentration of the standard, rboIFN-αI.1, that
and 25 µg\ml propidium iodide), resulting in the release of nuclei by corresponded to the same biological activity as measured for the samples
(meanpSD, n l 4).
nonadherent cells. The lysate was then added to the plate to release the
nuclei of the remaining adherent cells. Nuclei were analysed with a
FACScan flow cytometer (Becton Dickinson) after 30 min incubation at
4 mC in the dark. A minimum of 10% nuclei per sample was analysed. supernatants ranged from 13 to 55 % depending on the virus

Virus titration. Viruses were passaged and titrated in bovine strain used (Fig. 3). In comparison, a dose of 2000 pg\ml
turbinate cells as described previously (Adler et al., 1994) and the titres of recombinant bovine IFN-αI.1 primed for 15 % (Fig. 4 b) and a
the virus stocks and supernatants were calculated according to Reed & dose of 20 000 pg\ml primed for only 26 % (result not shown).
Muench (1938). Priming for activation-induced apoptosis was also observed
with supernatants of Mφ infected with BHV-1. These super-
natants were markedly more potent in priming for apoptosis
Results than those of Mφ infected with the NADL strain, followed by
Induction of IFN the TGAC and SuwaCP cp BVD virus strains (Fig. 3), which
The TGAC strain of BVD virus was shown to induce IFN parallels the capacity of these strains to induce IFN type I in
type I in bone marrow-derived Mφ (Adler et al., 1997). In the Mφ (Fig. 1). However, the extent of priming for activation-
present study, this effect was also observed in bovine induced apoptosis could not be explained by their antiviral, i.e.
monocyte-derived Mφ infected with the cp TGAC, SuwaCP IFN-like, activity.
and NADL strains of BVD virus. Taking rboIFN-αI.1 as the
standard, the mean concentration of IFN was found to range
between 130 and 400 pg\ml, depending on the virus strain Dissociation of priming for apoptosis from IFN type I
used to infect Mφ (Fig. 1). No antiviral activity was detected in activity
supernatants of Mφ infected with ncp BVD virus (TGAN, IFN type I produced by monocyte-derived Mφ in response
SuwaNCP) or in those of uninfected Mφ. However, the to virus infection consists of a mixture of the IFN type I
concentrations of IFN type I induced by cp BVD virus were subfamilies (Ohmann et al., 1987 ; Sager et al., 1998) and,
rather low in comparison with those induced by infection with therefore, its effect on target cells may differ from that of
BHV-1, which were two to three orders of magnitude higher rboIFN-αI.1 at equivalent concentrations. To obtain more
(Fig. 1). direct information on the role of IFN present in supernatants of
infected Mφ in priming for activation-induced apoptosis, we
investigated the effect of a soluble receptor of IFN type I
Activation-induced apoptosis encoded by the B18R gene of vaccinia virus (Alcamı! & Smith,
Inactivated supernatants of cp BVD virus-infected Mφ were 1992). B18R dramatically decreased the antiviral activity of
capable of priming uninfected Mφ for activation-induced IFN type I, regardless of whether it was due to rboIFN or
apoptosis in response to LPS as quantified by the appearance of natural bovine IFN type I produced by Mφ in response to
subgenomic DNA (Figs 2 and 3). The activity that primed for infection with cp BVD virus or BHV-1 (Fig. 4 a). Accordingly,
LPS-induced apoptosis was clearly restricted to supernatants of B18R inhibited priming for LPS-triggered apoptosis by
cp BVD virus-infected Mφ (NADL, SuwaCP, TGAC). In- rboIFN-αI.1. In contrast, the soluble IFN receptor B18R had no
duction of apoptosis was not observed with supernatants of effect on priming for activation-induced apoptosis by super-
ncp BVD virus-infected Mφ (SuwaNCP, TGAN). The per- natants of Mφ infected with cp BVD viruses (Fig. 4 b). The
centage of nuclei with subgenomic DNA induced by undiluted soluble IFN receptor partially reduced priming by supernatants

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L. Perler and others

(a)

(b)

Fig. 4. Dissociation of antiviral and apoptosis-priming activity. RboIFN-αI.1


(500 or 2000 pg/ml) and inactivated supernatants (filled bars) from cp
BVD virus- (NADL, TGAC, SuwaCP), ncp BVD virus- (TGAN, SuwaNCP),
BHV-1- or mock-infected Mφ were incubated either with a soluble
receptor for IFN type I (B18R, open bars) or with similarly treated product
from the empty vector (shaded bars). Supernatants of BHV-1-infected Mφ
contained high concentrations of IFN type I and were, therefore, diluted
1 : 1000. (a) The remaining biological activity of IFN type I was measured
as described in Fig. 1. IFN type I is expressed in concentrations of the
standard, rboIFN-αI.1 (meanpSD, n l 4). (b) The priming activity for
activation-induced apoptosis in response to LPS was analysed by flow
cytometry as described in Fig. 2 (meanpSD, n l 4).
Fig. 2. Flow-cytometric analysis of activation-induced apoptosis. Bovine
monocyte-derived Mφ were incubated with supernatants of TGAC cp BVD
virus-, TGAN ncp BVD virus- or mock-infected Mφ. After a second
incubation with LPS, DNA fragmentation was quantified by FACS analysis
of Mφ infected with BHV-1, which may be due to the relatively
as described in Methods. The FACS histograms show a representative high concentration of IFN present in these supernatants.
experiment, with propidium iodide fluorescence detected in FL1 (x-axis)
and the number of nuclei given on the y-axis.
Characteristics of the priming activity
A set of experiments was performed to characterize further
the factor(s) contained in supernatants of cp BVD virus-
infected Mφ responsible for priming for activation-induced
apoptosis. The molecular mass was estimated by ultrafiltration
of supernatants. Whereas filtration with membranes of an
exclusion size of 100 kDa had no effect on the priming activity,
a filter with a nominal exclusion size of 50 kDa eliminated the
activity (not shown). Since many proteins with molecular
masses of about 30 kDa are retained by the 50 kDa cut-off
filters (Amicon), the molecular mass is between approximately
Fig. 3. Activation-induced apoptosis. Bovine monocyte-derived Mφ were
incubated with inactivated supernatants of cp BVD virus- (NADL, TGAC,
30 and 100 kDa. The priming activity of the supernatants was
SuwaCP), ncp BVD virus- (TGAN, SuwaNCP), BHV-1- (diluted 1 : 1000) or stable when exposed to pH 2 at 4 mC for 24 h, resisted heating
mock-infected Mφ. Forty-eight h after stimulation with LPS, DNA at 56 mC for 30 min but not boiling for 15 min (Adler et al.,
fragmentation was quantified by FACS analysis as described in Fig. 2. The
y-axis indicates the percentage of nuclei with subgenomic DNA
1997). Significant differences in the virus load between the two
(meanpSD, n l 4). BVD virus biotypes may influence the release of the apoptosis-

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Virus-induced factors prime for apoptosis

priming factor(s). Distribution of cell-free and cell-associated bone marrow-derived cultures of Mφ infected in a similar
virus of cp and ncp BVD virus grown in Mφ may affect the manner (M. Schweizer, unpublished observation). This sug-
infected cell differentially. Titration of either cell-free or cell- gests that the Mφ-derived priming factor is distinct from
associated cp (SuwaCP) and ncp (SuwaNCP) BVD virus grown IFN-γ. Moreover, virus infection with both cp and ncp BVD
in Mφ revealed no significant differences between the two viruses failed to induce detectable amounts of TNF-α, as
biotypes. These virus strains, which are a virus pair, grew in determined by bioassay (Adler et al., 1996). This argues against
Mφ to titres of 10% to 10& TCID \ml. an important role of this cytokine in priming for activation-
&!
induced apoptosis.
It remains to be shown whether the Mφ is the only cell type
Discussion that produces a factor(s) capable of priming for apoptosis, and
The experiments reported here demonstrate that (i) in whether it is the only cell capable of being primed for
monocyte-derived Mφ, a range of cp, but not ncp, strains of activation-induced apoptosis. However, bovine turbinate cells
BVD virus induce the production of a factor(s) capable of produced neither IFN type I nor the priming factor in response
priming uninfected Mφ for activation-induced apoptosis ; (ii) to BVD virus or BHV-1 infection, nor could they be primed for
BHV-1 also induces such priming activity ; and (iii) priming by LPS-induced apoptosis (results not shown). Recently, Lambot
supernatants of infected Mφ is due entirely (BVD virus) or and co-workers showed that only the cp biotype of BVD virus
largely (BHV-1) to factors distinct from IFN type I. The induced apoptosis in bovine peripheral mononuclear cells.
conclusion that IFN type I was of no, or only minor, importance Directly relevant to this study is their observation that
for priming was reached, on the one hand, on the basis of a apoptosis in CD4+ and CD8+ T cells was enhanced sig-
comparison between dose–response curves of antiviral and nificantly by the presence of monocytes (Lambot et al., 1998).
apoptosis-inducing activities and, on the other hand, on the Together with the observations reported for BHV-1 (Hanon et
failure of a soluble receptor of IFN type I to block apoptosis, al., 1996 ; this work), HIV (Finkel et al., 1995), FIV (Mizuno et
despite its neutralization of antiviral activity. al., 1997) and murine cytomegalovirus (Koga et al., 1994), this
IFN type I was initially considered to be responsible for suggests that, during virus infection, mononuclear cells may
priming for apoptosis because, in infected Mφ, only BHV-1 play a role in regulating apoptosis in an array of other cell
and cp, but not ncp, BVD viruses induced IFN type I, which, in types. The concept that apoptosis of uninfected cells may
its recombinant form, was shown to prime uninfected Mφ for require at least two signals, i.e. one for priming for and one for
activation-induced apoptosis (Adler et al., 1995, 1997). How- triggering apoptosis, opens up new perspectives regarding the
ever, the mechanism of induction of IFN type I and of the study of the biochemistry involved.
priming factor(s) may be similar, which may explain the It will be of particular interest to investigate whether
correlation between the ability of the different virus strains apoptosis controlled by mononuclear phagocytes is related to
used to induce the formation of IFN type I and of the factor(s) lymphopoenia, a hallmark of most systemic virus infections. In
that primes for LPS-induced apoptosis (Figs 1 and 3). addition, the increased susceptibility of cattle infected by
Only a few viruses, HIV (Finkel et al., 1995 ; Cottrez et al., BHV-1 to secondary bacterial infections, e.g. Pasteurella spp.
1997 ; Chen et al., 1998 ; Herbein et al., 1998), murine (Bielefeldt Ohmann & Babiuk, 1985), may be related to
cytomegalovirus (Koga et al., 1994) and feline immuno- activation-induced cell death. Moreover, ‘ priming ’ may not be
deficiency virus (FIV) (Mizuno et al., 1997), have been reported restricted to mediating sensitivity to activation-induced apop-
to date to prime uninfected cells for activation-induced tosis, but may include other functional changes in uninfected
apoptosis. The priming for activation-induced apoptosis seems cells. Priming by endogenously produced IFN type I and by
to be due to different mechanisms. HIV has been shown to exogenously added IFN-αβ down-regulated IFN-γ plus TNF-α-
prime T cell apoptosis by a factor(s) released from monocytes induced nitric oxide production in mouse Mφ infected by tick-
(Chen et al., 1998), but direct contact between infected and borne encephalitis virus and in uninfected Mφ, respectively
uninfected cells may also be involved (Cottrez et al., 1997). The (Kreil & Eibl, 1995). The immunosuppression observed during
observations made in FIV infection point to soluble mediators measles is another example of such functional changes. It seems
released from infected cells. The factor involved has not been remarkable that an as yet unidentified factor(s) released from
characterized in any of these studies. Several studies have one B lymphocyte sufficed to impair antigen presentation by
attributed a role of soluble factors in priming for apoptosis to some 120 B lymphocytes (Fujinami et al., 1998 ; Sun et al.,
certain cytokines, among them IL-4 (Mangan et al., 1992) and 1998). These findings suggest that ‘ priming ’ may play an
IFN-γ (Munn et al., 1995 ; Bingisser et al., 1996). This raised the important role in the pathogenesis of virus infections.
question as to whether a previously described cytokine
mediates priming. IFN-γ had to be considered, since in some of This work was supported by the Swiss National Science Foundation,
the blood-derived cultures both IFN-γ mRNA and bioactivity grant F 31-50745.97. The authors wish to thank Dr G. L. Smith of the
were detected. However, this signal was tentatively attributed University of Oxford, UK, for his generous gift of B18R plasmid, Dr
to lymphocytes rather than to Mφ, since it was not detected in Marc Strasser for the isolation and Hanspeter Stalder for his help with the

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L. Perler and others

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her help with the preparation of macrophage cultures and Ruth Parham inducible enzyme RNase L causes apoptosis of animal cells. Virology 236,
for linguistic improvements. 354–363.
Evan, G. & Littlewood, T. (1998). A matter of life and cell death. Science
281, 1317–1322.
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