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J Virol, March 1998, p. 1853-1861, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Lymphocyte Apoptosis during Classical Swine Fever:
Implication of Activation-Induced Cell Death
Artur
Summerfield,*
Sonja M.
Knötig, and
Kenneth C.
McCullough
Institute of Virology and Immunoprophylaxis,
CH-3147 Mittelhäusern, Switzerland
Received 14 October 1997/Accepted 14 November 1997
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ABSTRACT |
Infection of pigs with classical swine fever virus (CSFV), a member
of the Flaviviridae family, causes a severe leukopenia, particularly notable with the lymphocytes. The goal of this study was
to analyze mechanisms behind this CSFV-induced lymphopenia. To this
end, the kinetics of leukocyte depletion, the appearance of apoptotic
cells, and virus infection of leukocytes after infection of pigs with
the virulent CSFV strain Brescia were analyzed. Depletion of B and T
lymphocytes was noted as early as 1 day postinfection (p.i.).
Circulating viable lymphocytes with reduced mitochondrial transmembrane
potential
a particular early marker for apoptosiswere also
detectable as early as 1 day p.i. When isolated peripheral blood
mononuclear cells were cultured for 6 h, significantly more sub-G1 cells with reduced DNA content were detected among
the lymphocytes from CSFV-infected animals, again as early as 1 to 3 days p.i. The first time virus was first found in the plasma, as well
as infection of leukocytes, was 3 days p.i. However, throughout the
observation time of 1 week, <3% of the circulating leukocytes and no
lymphocytes contained virus or viral antigen. Further analysis of the T
lymphocytes from infected animals demonstrated an increase in CD49d,
major histocompatibility complex class II, and Fas expression. An
increased susceptibility to apoptosis in vitro was also observed, particularly after addition of concanavalin A as well as
apoptosis-inducing anti-Fas antibody to the cultures. Taken together,
these results imply that activation-induced programmed cell death was
the mechanism behind lymphopenia during classical swine fever.
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INTRODUCTION |
Leukopenia can result in
immunosuppression and is a hallmark of certain virus infections, such
as classical swine fever (CSF), bovine viral diarrhea, and dengue
fever, all caused by virus members of the family
Flaviviridae (for reviews, see references 27, 54, and 55).
Classical swine fever virus (CSFV), a member of the genus
Pestivirus, is a small enveloped RNA virus causing an
economically important and fatal disease of pigs. The virus is known to
have a particular affinity for cells of the immune system, which seems to relate to the detrimental effects on the immune and hematopoietic systems (9, 41, 52, 54, 55). The target cells for CSFV in
the peripheral blood appear to be mainly monocytes, although in later
stages of the disease infection of lymphocytes (50, 55) as
well as granulocytic cells (50) has been noted. All leukocyte populations can be depleted during CSF, but B lymphocytes are
particularly sensitive (52). Despite this current knowledge, the immunopathological mechanisms and the role played by the virus infection of leukocytes with respect to the disease pathology in
general, and leukocyte death in particular, have not been elucidated. Generally, leukopenia could be a result of cell death, suppression of
hematopoiesis, or change in the distribution of leukocytes within
different compartments of the immune system. Leukocyte death can be
caused by necrosis or apoptosis. The latter, a suicide-like and
genetically programmed form of cellular death, is involved in
physiological as well as pathological cell death, induced by either a
lack or presence of particular stimuli (10). Late stages of
apoptosis are characterized by typical morphological criteria and
degradation of DNA (10). It has recently been reported that other characteristic cellular features, particularly a reduction in
mitochondrial transmembrane potential (
m),
precede these morphological and nuclear changes (26, 57).
Due to the important role played by apoptosis in the regulation of
leukocyte numbers (2, 3, 10, 25, 29), as well as the
observation that virus infections can be associated with apoptosis
(1, 40, 48), an important step in understanding the
pathogenesis of virus-induced leukopenia would be to determine the role
played by apoptosis in the depletion of lymphocytes.
In this study, we (i) analyzed the relationship between the kinetics of
leukocyte depletion in the peripheral blood and the virus infection
therein and (ii) determined the implication of apoptosis. A significant
reduction of lymphocyte numbers in the blood of CSFV-infected pigs was
noted as early as 1 day postinfection (p.i.), before viremia or
virus-infected leukocytes were apparent. An increase in lymphocytes
programmed to die by apoptosis was also observed early p.i., implying
an important role for apoptosis in the destruction of leukocytes during
this disease. We present data suggesting a mechanistic role for
Fas-mediated activation-induced cell death (AICD).
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MATERIALS AND METHODS |
Infection of pigs with CSFV.
A total of 18 animals (3 months
of age) were infected oronasally with the highly virulent CSFV strain
Brescia (kindly provided by H.-J. Thiel, University of Giessen,
Giessen, Germany; 12), using 106 50%
tissue culture infective doses (TCID50)/animal. Body
temperature and clinical symptoms were recorded daily. Before infection
(day 0) and on days 1, 3, 5, and 7 p.i., blood samples were taken. Some animals were slaughtered on days 1, 3, 5, and 7 p.i. for collection of tonsils.
Preparation of leukocytes.
Peripheral blood leukocytes (PBL)
were obtained by incubation of citrated blood in NH4Cl
buffer (0.15 M NH4Cl, 10 mM NaHCO3 [pH 7.4])
for 5 min at 4°C to lyse erythrocytes. For complete erythrocyte
lysis, this treatment was repeated three times, followed by a wash in
Ca2+-Mg2+-free phosphate-buffered saline
(PBS-A) supplemented with 0.035% (wt/vol) EDTA and centrifugation at
250 × g for 10 min at 4°C. Peripheral blood
mononuclear cells (PBMC) were isolated by Ficoll-Paque (1.077 g/liter)
density centrifugation from isolated buffy coats as described
previously (33). Contaminating erythrocytes were lysed by a
single treatment with NH4Cl buffer as described above. Tonsil cell suspensions were prepared by collagenase-DNase (Boehringer Mannheim) digestion as described previously (19).
MAbs and two-color flow cytometric analysis (FCM).
For
phenotyping, the following monoclonal antibodies (MAbs) were used:
anti-SWC1 (11/8/1 [44]) and anti-major
histocompatibility complex (MHC) class II (MSA-3
[20]), both kindly contributed by A. Saalmüller
(Bundesforschungsaustalt für Viruskraukheiten der Tiere,
Tübingen, Germany), anti-SWC3 (74-22-15; American Type Culture
Collection [5]), anti-SWC8 (MIL-3; Serotec
[21, 44]), goat anti-pig surface immunoglobulin (sIg;
Jackson ImmunoResearch Laboratories), anti-CD3 (BB23-8E6; VMRD Inc.,
Pullman, Wash. [44]), anti-Fas (CH-11; Upstate
Biotechnology Inc., Lucerne, Switzerland), and anti-CD49d (HP2.1;
Immunotech, Marseille, France). Using an SWC3-SWC8
double-immunofluorescence analysis, we identified granulocytic cells as
SWC3+ SWC8+ and monocytes as SWC3+
SWC8
(21, 49). In SWC1-SWC3 double labelings
of PBL, SWC1+ SWC3 T and NK cells were
discriminated from SWC1 SWC3 B cells and
SWC1+ SWC3+ monocytes (43). Indirect
immunofluorescence labeling and acquisition of data on a FACScan
(Becton Dickinson) were done as previously described (49).
Virus infection detection by FCM.
For detection of
CSFV-infected cells, we used the anti-CSFV E2 MAb HC/TC26 (IgG2b;
kindly provided by Dr. Bommeli AG, Bern, Switzerland
[17]). For this labeling, the cells were fixed and permeabilized (Cell Permeabilisation kit; Harlan Sera-Lab, Crawley Down, England) according to the instructions for the kit before addition of MAb HC/TC26 for 15 min. Goat anti-mouse IgG2b-phycoerythrin was used as the detection antibody as described above.
Detection of virus infection in cell cultures and virus
titration.
PBMC prepared from CSFV-infected animals were cultured
at 106 cells/ml on monolayers of PK-15 indicator cells in
24-well plates. After 4 days of incubation, the indicator cells were
fixed with 70% ethanol (
20°C, 10 min), and immunofluorescence
detection of virus E2 glycoprotein by using MAb HC/TC26 was performed.
For determination of virus titer in plasma, samples were titrated on
PK-15 cells in 24-well plates (six replicates) and incubated for 1 h at 37°C. Then the inoculum was removed, and the cells were washed
and cultured for 72 h at 37°C. After immunofluorescence labeling
as described above, the culture was analyzed for infectious foci under
a UV microscope. The virus titers were calculated by the method of
Kaerber (23).
Cell viability and apoptosis analysis.
The carbocyanine dye
3,3'-dihexyloxacarbocyanine iodide (DiOC6[3]) was
used to determine m (38) on the
basis of the fact that this method detects cells at an early stage
during apoptosis (39, 57). To this end, 106
cells were incubated with 40 nM DiOC6[3] in PBS for 10 min at 37°C. DiOC6[3] fluorescence was detected in the
FL-1 channel (525 nm) of the flow cytometer. As negative controls,
cells were treated with the uncoupling agent carbonyl cyanide
m-chlorophenylhydrazone (50 µM; Sigma). To distinguish
early apoptotic cells, which show a low DiOC6[3]
staining, from dead cells, propidium iodide (PI) was added after
FL-1/FL-2 compensation.
For a method to quantify late stages of apoptosis, cells were fixed
with 75% (vol/vol) ethanol (4°C, 2 min), washed, and centrifuged. PI
(50 µg/ml plus 100 µg of RNase per ml) staining of DNA was effected
for 30 min at 37°C, and then cells were analyzed by FCM. The DNA
histograms obtained after labeling of the permeabilized cells with PI
were used to quantify apoptotic cells with reduced DNA, located in the
sub-G1 region (11, 36, 37).
Cell culture.
PBMC were cultured either in round-bottom
microtiter plates, for proliferation assays, or in 24-well plates, for
cell death measurements, at a concentration of 106 cells/ml
in RPMI 1640 supplemented with 2 mM L-glutamine, 5 × 105 M 2-mercaptoethanol, 10 mM HEPES buffer, and 10%
fetal calf serum. In certain experiments, cells were stimulated with
concanavalinA (ConA; 10 µg/ml) or human recombinant interleukin-2
(IL-2; 50 U/ml; Boehringer Mannheim). Cell proliferation was measured
after 72 h by incubating the cells with 1 µCi of
[3H]thymidine/well for an additional 18 h. After
harvesting on glass fiber filters, counts per minute were read with a
Trace 96 counter (Inotech, Dottikon, Switzerland).
Microscopic analysis of lymphocytes.
Cells were
cytocentrifuged onto glass microscope slides, stained by using a
Diff-Quik staining kit (Baxter Diagnostik AG, Düdingen,
Switzerland), and analyzed by light microscopy. Analysis of nuclear
changes occurring in late apoptotic stages was also performed on
ethanol-fixed cells, stained with PI as described above, under a UV
microscope. Leukocyte counts were performed with heparinized blood
after lysis of erythrocytes in Türk's solution.
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RESULTS |
Kinetic analysis of PBL populations during acute CSF.
Acute
CSF is characterized by a rapid development of fever, with the typical
hematological feature being leukopenia (24, 52, 54, 55).
After infection of pigs with CSFV strain Brescia, the majority of the
animals had elevated body temperatures (>40°C) by day 3 p.i.;
this further increased, reaching the highest levels at 4 days p.i.
(Fig. 1A). In addition, a rapid onset of
leukopenia was seen. By 2 days p.i., the number of PBL of all 18 infected animals had dropped to under 10,000 cells/µl of blood (Fig.
1B). In healthy sex- and age-matched pigs from the
specific-pathogen-free breeding unit of our institute, the average was
13,100 (±2,400) leukocytes/µl (24 pigs analyzed). This loss of PBL
further progressed, by day 5 p.i., reaching levels as low as 2,100 to 5,350 cells/µl.
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FIG. 1.
Body temperatures and PBL counts following infection of
pigs with CSFV strain Brescia (results for six representative animals).
(A) Change in body temperature over time; (B) PBL counts of the animals
determined by microscopic counting of leukocytes in heparinized
whole-blood samples, after lysis of erythrocytes with Türk's
solution.
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By phenotyping PBL from infected pigs on days 0, 1, 3, 5, and 7 p.i., both the relative and absolute amounts of the different
PBL
populations were determined. Myeloid cells, including both
granulocytic
cells and monocytes, were defined as SWC3
+ cells
(
21); T lymphocytes and NK cells were identified as
SWC1
+ SWC3
cells, and B cells were identified
as sIg
+ SWC1
cells (
43). The
results revealed that during the onset of leukopenia,
mainly the
lymphocyte but not myeloid populations were depleted
(Fig.
2). As early as 1 day p.i., an
approximately twofold drop
in the number of T and B lymphocytes was
seen (Fig.
2A and B),
whereas myeloid cells remained more stable, with
some degree of
variation between animals (Fig.
2C). By day 3 p.i.,
the number
of B and T lymphocytes had further decreased at least
threefold,
reaching their lowest levels by 5 days p.i. (Fig.
2A and B).
At
this time point, less than 1/10 of the initial numbers of B and
T
cells were found in the blood.
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FIG. 2.
Kinetic analysis of the numbers of different leukocyte
populations in the first week of CSF (results for six representative
animals). PBL preparations were labeled with combinations of
antileukocyte MAbs, and the percentage of positive cells was determined
via analysis by immunofluorescence FCM. The absolute numbers of the
different populations were then calculated from the total PBL counts
obtained at each time point. (A) B cells, identified as surface
SWC1 Ig+; (B) T cells and NK cells,
identified as SWC1+ SWC3; (C) myeloid cells
composed of monocytic and granulocytic cells, identified as
SWC3+.
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Mitochondrial dysfunction in PBMC during CSF.
Apoptotic cells
are removed by phagocytosis in vivo (10, 14, 46), rendering
it difficult to detect apoptosis ex vivo in the peripheral blood
(15, 30, 32). Therefore, an early marker detecting cells
programmed to dieDiOC6[3], which measures the reduction
of mwas used.
The results in Fig.
3 show a
representative example of PBMC from an animal analyzed before infection
and 1, 3, 5 and 7 days
p.i. In the forward scatter (FSC)/side scatter
(SSC) plots shown
in the top row of Fig.
3, an increase in the number
of cells with
reduced size was observed (cells located left of region
G1); this
was most apparent from 3 days p.i. Furthermore, the
appearance
of cells with high SSC in the PBMC preparations was also
found
beginning at 3 days p.i. These cells had the morphology and
phenotype
of immature granulocytic cells (
50). When
m was measured
in these PBMC, a reduction
in the percentage of viable cells with
high
m was observed during the course of
infection
(Fig.
3, middle row, cells in R1). The
mlow cells showing early signs
of programmed cell death (PCD) (cells
in R2) and nonviable
PI
+ cells (cells in R3) increased following infection.
Cells with
reduced
m were located mainly
in the region with the
lowest FSC/SSC (data not shown). Consequently,
when the G
1 gate
of the lymphocyte region within the
FSC/SSC plot shown in the
top row of Fig.
3 was used to exclusively
analyze these cells,
the percentage of
mhigh cells was greater than
in ungated PBMC (Fig.
3, bottom row).
Nevertheless, within this gate
the percentage of cells with reduced
m
(R2) increased fourfold.
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FIG. 3.
Reduction of m and
increased cell death in circulating PBMC during acute CSF (results for
a representative pig). For determination of
m, freshly isolated
DiOC6[3]-labeled PBMC obtained from pigs before infection
(n.i.) and 1, 3, 5, and 7 days p.i. (d.p.i.) were analyzed by FCM. The
upper row shows the FSC/SSC plots, and the two lower rows show the
DiOC6[3]-PI staining of the cells. A region in the
FSC/SSC plots was used to define a gate (G1) for the exclusive analysis
of DiOC6[3]-PI staining of cells with scatter
characteristics typical of viable lymphocytes. This is shown in the
bottom row of contour plots (gate R1). Regions R1 to R3 defined in the
DiOC6[3]-PI contour plots were used determine the number
of DiOC6[3]high PI (R1),
DiOC6[3]low PI (R2), and
DiOC6[3]low PI+ (R3) cells.
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In Fig.
4, the results of the analyses
exemplified in Fig.
3 are shown for all pigs. The reduction of the
viable population
with high
m was detected
as early as 1 to 3 days p.i.,
reaching the lowest levels from day
3 p.i. on (Fig.
4A). In addition
to the reduction of the healthy
cells with high
m,
we noted an increase of
dead PI
+ cells during CSF, from 4 to 6% of the PBMC before
infection to
over 10% by day 5 p.i. (Fig.
4B).
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FIG. 4.
Reduced m and increased
cell death of PBMC following infection of pigs with CSF.
m and cell membrane permeability (cell
death) were measured as described for Fig. 3. Following infection of
pigs with CSFV, changes in the percentage of viable PBMC
(DiOC6[3]high and PI) (A) and
the percentage of PI+ dead PBMC (B) were determined. The
averages and standard deviations for six (for days 0, 1, and 3 p.i.) and three (for days 5 and 7 p.i.) different pigs were
calculated.
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Apoptosis of peripheral leukocytes during CSF.
To confirm that
the viable cells with reduced m in the
PBMC preparations from CSFV-infected animals would relate to apoptotic
cells, an additional method characteristically employed for identifying
PCD was used for analysis of the samples. This method was cell cycle
analysis to determine the percentage of cells with reduced DNA content
in the sub-G1 region that were known to be apoptotic
(11, 36, 37). PBMC from noninfected pigs gave values of less
than 1% for the sub-G1 region (Fig.
5A). In Fig. 5B, PBMC from a pig at 7 days p.i. show the presence of 6.1% of the cells in the
sub-G1 region. The first PBMC preparations containing
detectable apoptotic sub-G1 cells were obtained 3 days p.i.
(Fig. 5C): two of six animals analyzed at this time point clearly
contained sub-G1 cells. One animal of the three analyzed at
5 days p.i., and all three animals analyzed at 6 days p.i., had
elevated numbers of cells with reduced DNA content (Fig. 5C).
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FIG. 5.
DNA loss in leukocytes during CSF. PBMC obtained from
animals before and after infection with CSFV were analyzed for the
percentage of cells located in the sub-G1 region of a cell
cycle analysis by FCM. The FSC (x axis) versus the
fluorescence intensity (y axis) of PI-stained ethanol-fixed
cells (representing a measurement of DNA content) is illustrated in
contour plots (A, B, D, and E), with the cell cycle stage indicated to
the right of plot B. (A and B) Examples of results obtained with cells
from a pig before infection (A) and 7 days p.i. (B); (C) percentage of
PBMC located in the sub-G1 region of all individual animals
analyzed with respect to time p.i.; (D to F) equivalent results for the
same cells but after culture for 6 h at 37°C.
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Previous work has demonstrated the difficulties with detecting
apoptotic cells which had arisen in vivo by methods based on
DNA
fragmentation (
15,
32,
57). Such difficulties would
relate
to the results obtained and shown in Fig.
5C. Consequently,
the
isolated PBMC were cultured for 6 h to permit cells committed
to
PCD to develop signs of DNA fragmentation and loss, as determined
by
the DNA content analysis. As demonstrated in Fig.
5D to F,
the levels
of sub-G
1 cells in PBMC from noninfected animals remained
below 2%. In contrast, PBMC preparations from four of six animals
obtained at 1 day p.i., and all PBMC preparations obtained from
days 3 to 7 p.i., contained significantly increased levels of
sub-G
1 apoptotic cells, compared to the PBMC from
noninfected
animals (Fig.
5F). Examples of the staining pattern
obtained are
shown in Fig.
5D (noninfected pig) and 5E (cells from a
typical
animal at 7 days p.i.). Morphological analysis of cytospin
preparations
of these PI-stained cells under a UV microscope confirmed
the
presence of these apoptotic cells with condensed DNA, nuclear
fragmentation, and/or DNA-containing apoptotic bodies (data not
shown).
Viremia and virus infection of leukocytes.
Due to the capacity
of CSFV to infect different leukocyte populations (9, 50, 54,
55), the presence of virus in plasma and leukocytes was
determined. The objective was to elucidate what relationship, if any,
might exist with the rapid lymphopenia shown above. The first
detectable presence of infectious virus in the plasma was at 3 days
p.i. (Table 1). Virus titers in the plasma did not significantly increase before day 5 p.i., reaching levels between 103 and 105
TCID50/ml.
When PBL and PBMC were tested for viral glycoprotein E2 expression by
FCM, the first positive cells were detectable at 3 days
p.i. for
certain animals and 5 days p.i. for the majority of animals.
Nevertheless, the numbers of infected cells was always low (Table
1).
Even at day 7 p.i., not more than 2% of the PBMC were
E2
+. In double-labeling experiments, these cells were shown
to be
SWC3
+ and thus of myeloid origin (data not shown).
Tonsillar samples
had detectable virus-infected cells as early as 1 day
p.i. but
not more than 1% and never more than 12%. When PBMC were
cocultured
with CSFV-permissive indicator cells (PK-15) for 5 days, it
was
possible to detect infected cells as early as 3 days p.i., but
only
in three of six animals analyzed; all animals were positive
by day
5 p.i. (Table
1).
Modulation of T-lymphocyte phenotype and Fas expression.
The
observation that no infection of lymphocytes by CSFV was detectable
during the 7 days of observation, and that CSFV is not cytopathogenic
for lymphocytes in vitro (reference 55 and data not
shown), suggests that virus infection was not a direct cause of
lymphocyte death. When the phenotype of lymphocytes from infected pigs
was analyzed, an increased expression of CD49d and MHC class II was
observed, as shown for a representative pig in Fig.
6 (top and middle rows). Before infection
with CSFV, 21 to 33% of T lymphocytes from the different pigs
expressed high levels of CD49d (exemplified by the representative
samples shown in the top row of Fig. 6, column n.i.). This number
increased for all pigs analyzed 3 days p.i. and reached maximum levels
by 5 days p.i. when 76 to 87% of T cells were CD49dhigh.
Interestingly, an increase in the FSC, especially of the
CD49dhigh T lymphocytes, was noted, indicating an increase
in activated cells (Fig. 6, top row). Concerning the MHC class II
expression on T lymphocytes from CSFV-infected pigs, MHC class
II+ T lymphocytes increased from 6 to 15% before infection
to 70 to 80% by 5 days p.i. The cells with increased FSC were mainly among the MHC class II+ population.
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FIG. 6.
Increased expression of CD49d, MHC class II, and Fas on
peripheral T lymphocytes during CSF (results for a representative
animal). PBMC obtained from pigs before infection (n.i.) and 1, 3, 5, and 7 days p.i. (d.p.i.) were immunofluorescence labeled for CD3 versus
CD49d, CD3 versus MHC class II, and CD3 versus Fas expression. The
expression of CD49d (top row) and MHC class II (middle row) on gated
CD3+ cells is shown in FSC (x axis) versus
immunofluorescence (y axis) contour plots. The expression of
Fas on CD3+ cells is shown in histograms (bottom row).
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Under certain circumstances, activation of T lymphocytes can result in
apoptotic death mediated by Fas (CD95)-Fas ligand (FasL)
interaction, a
process termed AICD (
4,
22,
25,
29,
42).
The possible
implication of AICD in T lymphocytes during CSF was
analyzed in terms
of Fas expression on the T cells. As shown for
a representative pig in
Fig.
6 (bottom row), an increase in the
number of Fas-expressing T
lymphocytes was observed during the
course of the disease. Before
infection, 6 to 17% of T cells from
the different pigs expressed Fas,
and this number increased to
reach over 30% for most pigs from 3 to 5 days p.i.
Impaired responsiveness of T lymphocytes to mitogen stimulation
during CSF.
The lymphoid depletion and phenotypic changes of T
lymphocytes described above could have been the result of migratory
changes within the T-lymphocyte compartments. If redistribution of
lymphocytes alone were responsible for the lymphopenia during CSF, an
intact functional activity of the remaining circulating T cells would be expected. To investigate this possibility, we analyzed the proliferative capacity of PBMC obtained before and after infection with
CSFV strain Brescia. A four- to fivefold depression of the T-lymphocyte
response to ConA was found 3 days p.i.; this low level was maintained
until the end of the experiment (Fig.
7B). This finding is in agreement with
previous reports describing a persistent decreased proliferative
response of peripheral lymphocytes to mitogens from day 2 p.i.
with virulent CSFV (56). Interestingly, the proliferative
response to IL-2 was not impaired (Fig. 7C). In fact, PBMC from certain
animals, particularly 5 and 7 days p.i., showed enhanced proliferation
upon treatment with IL-2.
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FIG. 7.
Changes in the proliferative capacity of PBMC during
CSFV infection. PBMC obtained from pigs before infection (0) and 1, 3, 5, and 7 days p.i. (x axis) were culture for 3 days and an
additional 18 h for [3H]thymidine labeling to
determine proliferation. Due to the relative increase of myeloid cell
populations in the PBMC obtained from infected pigs (data not shown),
the cell number used was adjusted to obtain an equal concentration of
106 lymphocytes/ml in each culture. (A) Spontaneous
proliferation; (B) response to ConA; (C) response to IL-2. Each bar
represents the average of triplicates from the PBMC of an individual
pig.
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Culture-, ConA-, and Fas-induced apoptosis of lymphocytes from
CSFV-infected animals.
To determine the relationship between cell
death and reduced responsiveness to mitogen, PBMC from animals before
and after CSFV infection were treated with ConA for 24 h, and the
number of apoptotic sub-G1 cells was quantified. Cells from
infected pigs as early as 1 day p.i. showed enhanced susceptibility to apoptosis in the absence of ConA stimulation (Fig. 5F and
8). Addition of ConA increased the number
of dead cells obtained from both noninfected and infected pigs (Fig.
8). Similar effects were obtained by addition of anti-Fas antibody,
indicating that these cells were susceptible to Fas-induced cell death.
Interestingly, IL-2 was incapable of rescuing the cells from cell
death: the number of apoptotic cells remained at the level of the
culture without IL-2 or was even higher (data not shown).
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FIG. 8.
In vitro culture, ConA (10 µg/ml) treatment, and
anti-Fas (CH-11; 0.5 µg/ml) treatment result in enhanced apoptosis of
PBMC from CSFV-infected pigs. The same PBMC used for Fig. 7 were
cultured for 24 h, and apoptotic cells located in the
sub-G1 region were quantified by DNA content analysis. For
each time point, results for three representative pigs are shown.
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DISCUSSION |
Previous studies on the development of leukopenia during CSF have
tended not to analyze the early phase of the disease, yet the
characteristics of the leukopenia suggest that the primary initiating
pathogenic events are more likely to be elucidated when early phases
are focused on. In the present study, the induction of leukopenia
during acute CSF developed rapidly following infection with the
virulent Brescia strain. When focusing on the absolute counts of
lymphocytes, we noted a peracute depletion of this population as early
as 24 h p.i. At this time point, the infection was otherwise asymptomatic, and neither cell-free nor cell-associated virus could be
detected in the blood. Although infected cells could be identified in
the tonsils, it was not until 3 days p.i. with certain animals, and 5 days p.i. with all animals, that such cells were found in the blood.
Nevertheless, the numbers of infected cells were always very low, and
no virus antigen was detected in the lymphocytes. These results
indicate that the lymphopenia was not a direct effect of virus
infection in the lymphocytes. This conclusion is supported by the
observation that CSFV strain Brescia has no cytopathogenic effect on
lymphocytes in vitro (data not shown).
In contrast to the report of Susa et al. (52), no
significant difference in depletion between T and B lymphocytes was
found during the early phase of CSF, after infection with the Brescia strain. A possible explanation could be the higher virulence of the
Brescia strain than of the Alfort strain used by Susa et al. (52), resulting in a more profound effect on both T and B
cells. Furthermore, the use of absolute cell counts in the present
study, in contrast to the work of Susa et al. (52), was
required to compare the different rates of T- and B-lymphocyte
depletion.
Further insight into the possible mechanisms operating during the early
phase of lymphopenia was obtained by analysis of certain parameters for
cell viability and PCD. As early as 1 day p.i., we detected an
increasing number of lymphocytes with reduced
m, as well as cells programmed to die
(determined by a short culture period before DNA content analysis).
Such results point to PCD being involved in the early lymphocyte
depletion during CSF. The reduction of m
has been reported as an early event during apoptotic cell death
occurring before DNA fragmentation (7, 8, 26, 39, 57); it
also appears before expression of phagocytosis receptors such as
phosphatidylserine (7), crucial for the clearance of
apoptotic cells in vivo by phagocytes (14). This
m measurement makes it possible to detect
PCD in vivo, without concern that such cells had been removed by
phagocytosis and also without any additional cell culture. Macho et al.
have used the same principle to measure mitochondrial dysfunction of PBMC obtained from human immunodeficiency virus-infected patients (30). In addition to the leukocytes with a
m reduction, and the appearance of cells
programmed to die, an increasing number of dead PBMC was found during
the course of CSF. Whether all of these cells died from apoptosis
cannot be definitively said, since the end stages of apoptosis and
necrosis result in similar patterns of membrane permeabilization.
Taken together, these results relate to the observations of Korn and
Lorenz, who found an increase of necrotic cells and monocytes with
ingested lymphocytes in the blood of pigs infected with virulent CSFV
(24). Histological studies of lymphoid tissues from
CSFV-infected pigs also identified necrotic as well as phagocytosed
lymphocytes (9, 41); the latter would be indicative of
apoptotic lymphocyte death in these organs. Indeed, analysis of the
tonsils from infected pigs in the present study revealed a high degree
of apoptotic cell death in these organs at 5 to 7 days p.i. (data not
shown).
Because CSFV is noncytopathogenic, and given the observation that no
peripheral lymphocytes were infected but lymphocyte depletion was a
very early event, mechanisms other than a direct induction of cell
death by the virus had to be considered. One possible explanation comes
from the study of Bruschke et al. wherein a recombinant CSFV
glycoprotein Erns (E0) had a cytotoxic effect on
T cells in vitro (6). Erns is known
to be secreted from CSFV-infected cells and is found in the sera of
infected pigs (6). Nevertheless, the sera from neither
infected pigs nor infected pigs nor infected cell culture supernatants
were found to have a detrimental effect on lymphocytes from noninfected
pigs (data not shown). To investigate further the possible mechanisms
of lymphocyte depletion during CSF, a phenotypic analysis was
performed. The results obtained, namely, upregulation of CD49d and MHC
class II as well as increased T-lymphocyte size, were indicative of
T-lymphocyte activation and/or conversion to memory phenotype early
during the disease. CD49d has been shown to have a heterogeneous
expression on T lymphocytes, with memory cells and activated T
lymphocytes being CD49dhigh (31). In the pig,
some T lymphocytes express MHC class II constitutively, but upon
activation, MHC class II is upregulated (45). This upregulation of MHC class II on T cells is not necessarily a sign of
activationporcine memory Th lymphocytes are MHC class II+
even in a nonactivated stage (45, 51). With the
CSFV-infected animals, the kinetics of MHC class II increase correlated
with that of CD49d on the T-cell population. Consequently, the
increased MHC class II expression confirmed the CD49d results, showing
an increase in T lymphocytes with a memory/activated phenotype.
Interestingly, on most of the T lymphocytes, only a slight increase of
the IL-2 receptor (CD25) expression was observed; the majority of the
CD49dhigh MHC class II+ T lymphocytes
were actually CD25 (data not shown). Taken together
with the increased expression of Fas on the lymphocytes, these results
indicate that AICD could be involved in the induction of apoptosis in
lymphocytes during CSF. This would be in agreement with the current
concept that activated and memory T cells have increased Fas expression
(34) and reduced Bcl-2 expression (1, 3) and thus
show an increased susceptibility to apoptosis mediated by Fas-FasL
interaction (1, 3, 13, 22, 25). The physiological role of
this system is thought to be in the prevention of overstimulation of T
lymphocytes during the immune response (2), downregulation
of the immune response (1), and maintenance of lymphocyte
homeostasis (29).
The possible role of these mechanisms in the induction of lymphopenia
during CSF was confirmed by in vitro culture of T lymphocytes from
infected pigs and addition of apoptosis-inducing anti-Fas MAb. This
increased sensitivity to apoptosis in the presence of mitogen explains
the lack of proliferative response to this stimulus and relates to
AICD. ConA activation has been reported to increase cell death through
AICD, when the situation results in aberrant stimulation of the T cells
(42). Reduced mitogen responsiveness and increased
susceptibility to apoptosis, both associated with the activated
phenotype, have been reported for other viral infections (16, 18,
35, 40). IL-2 did not have the capacity to prevent apoptosis of T
cells from infected pigs when tested in vitro. In contrast, it
sometimes enhanced cell death, which again relates to other viral
infections where AICD has been implicated (40). Therefore, a
possible concept for lymphocyte depletion during CSF would be an
aberrant triggering of lymphocytes by virus-induced cytokine imbalance
or by a viral superantigen, resulting in activation, upregulation of
Fas, and downregulation of Bcl-2 (10, 40, 47). Fas-FasL
killing would be mediated by autocrine T-cell suicide (13,
53) or by interaction with FasL-expressing phagocytes (28), suggesting a dysregulation of the regulatory
Fas-dependent systems for T-lymphocyte homeostasis (25, 29),
which may be common to other viral infections (1, 3, 18, 35,
40). Similar mechanisms might also be responsible for the
B-lymphocyte depletion during CSF: Fas expression was also increased on
these cells (data not shown). However, this requires further
investigation, because the regulation of B-lymphocyte homeostasis
differs from that of T-lymphocyte homeostasis (47).
Further studies will now concentrate on the triggers of this cell death
and the localization of these pathogenic events. The tonsils and spleen
were also affected by apoptotic cell depletion, indicating that the
observed effects did become generalized throughout the lymphoid tissue.
Analysis of the functional alterations induced by virus infection in
the primary target cells in the lymphoid tissuemacrophages,
endothelial cells, dendritic cells, and probably other accessory cells
of the immune system (9, 52, 54, 55)should further
elucidate CSF immunopathogenesis.
| |
ACKNOWLEDGMENTS |
This work was supported by the Swiss Federal Veterinary Office.
We thank René Schaffner, Annette Arriens, Robert Tschudin, Heidi
Gerber, and Sandra Wenger for technical assistance and Christian Griot
for reviewing the manuscript. For animal care, we acknowledge the work
of Peter Zulliger, Markus Mader, and Andreas Michel.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Virology and Immunoprophylaxis, Sensemattstrasse 293, 3147 Mittelhäusern, Switzerland. Phone: 41-31-8489377. Fax:
41-31-8489222. E-mail: artur.summerfield{at}ivi.admin.ch.
| |
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Abstract
Introduction
