Modulation of splenic macrophages, and swine leukocyte antigen (SLA) and viral antigen expression following African swine fever virus (ASFV) inoculation


Gonzalez Juarrero, M.; Lunney, J.K.; Sanchez Vizcaino, J.M.; Mebus, C.

Archives of Virology 123(1-2): 145-156

1992


Expression of viral and major histocompatibility complex (MHC) antigens and localization of T cells and macrophages was studied in frozen tissue sections of spleens taken from normal pigs or from pigs inoculated with highly virulent Lisbon 60 (L60), or with moderately virulent Dominican Republic 1978 (DR-II), African swine fever virus (ASFV) isolates. Splenic sections from L60 inoculated pigs exhibited a large decrease in macrophage staining, whereas DR-II infected animals appeared more intensely stained in the macrophage sheath arteries. Class I and class II MHC expression was decreased in spleens from pigs infected with either isolate at 3 day post inoculation (DPI). This was reversed in DR-II inoculated pigs at 4 DPI. Splenic tissue sections from L60 inoculated pigs exhibited only a marginal increase in SLA expression at a later time, 6 DPI. We suggest that the recovery of SLA expression during infection of pigs with ASFV is associated with survival or replacement of macrophages in the spleen leading to an effective immune response against the virus.

_Archives
Arch
Virol
(1992)
123:
145-156
Urology
©
Springer-Verlag
1992
Printed
in
Austria
Modulation
of
splenic
macrophages,
and
swine
leukocyte
antigen
(SLA)
and
viral
antigen
expression
following
African
swine
fever
virus
(ASFV)
inoculation
M.
Gonzalez-Juarrero
l
'
*,
J.
K.
Lunney
2
,
J.
M.
Sfinchez-Vizcaino
3
,
and
C.
Mebus
4
I
U.S.
Department
of
Agriculture,
Plum
Island
Animal
Disease
Center,
ARS,
Greenport,
New
York,
U.S.A.
2
U.S.
Department
of
Agriculture,
Helminthic
Diseases
Laboratory,
LPSI,
ARS,
Beltsville,
Maryland,
U.S.A.
3
Instituto
Nacional
de
Investigaciones
Agrarias,
Departamento
de
Virologia
Animal,
Madrid,
Spain
4
U.S.
Department
of
Agriculture,
Foreign
Animal
Disease
Diagnostic
Laboratory,
APHIS,
Greenport,
New
York,
U.S.A.
Accepted
July
19,
1991
Summary.
Expression
of
viral
and
major
histocompatibility
complex
(MHC)
antigens
and
localization
of
T
cells
and
macrophages
was
studied
in
frozen
tissue
sections
of
spleens
taken
from
normal
pigs
or
from
pigs
inoculated
with
highly
virulent
Lisbon
60
(L60),
or
with
moderately
virulent
Dominican
Re-
public
1978
(DR-II),
African
swine
fever
virus
(ASFV)
isolates.
Splenic
sections
from
L60
inoculated
pigs
exhibited
a
large
decrease
in
macrophage
staining,
whereas
DR-II
infected
animals
appeared
more
intensely
stained
in
the
mac-
rophage
sheath
arteries.
Class
I
and
class
II
MHC
expression
was
decreased
in
spleens
from
pigs
infected
with
either
isolate
at
3
day
post
inoculation
(DPI).
This
was
reversed
in
DR-II
inoculated
pigs
at
4
DPI.
Splenic
tissue
sections
from
L60
inoculated
pigs
exhibited
only
a
marginal
increase
in
SLA
expression
at
a
later
time,
6
DPI.
We
suggest
that
the
recovery
of
SLA
expression
during
infection
of
pigs
with
ASFV
is
associated with
survival
or
replacement
of
macrophages
in
the
spleen
leading
to
an
effective
immune
response
against
the
virus.
Introduction
African
swine
fever
virus
(ASFV)
is
an
icosahedral,
double-stranded
DNA,
enveloped
virus
classified
in
the
family
hidaviridae[1].
African
swine
fever
was
*
Current
address:
ILRAD,
Nairobi,
Kenya.
146
M.
Gonzalez-Juarrero
et
al.
originally
described
as
an
acute
and
contagious
infection
of
domestic
swine
with
mortality
approaching
100%
[2].
However,
during
the
spread
of
the
disease
from
Africa
to
Europe
and
America,
ASFV
has
developed
patterns
of
virulence
varying
from
peracute
to
chronic
to
asymptomatic
[3,
4].
The
porcine
mono-
cytic-phagocytic
system
has
been
identified
as
the
primary
target
cell
of
ASFV
[5].
Pigs
infected
with
the
highly
virulent
form
of
ASFV,
e.g.,
Lisbon
60
(L60)
or
Tengani,
usually
die
by
6-10
days
post
inoculation
(DPI)
with
characteristic
lesions
of
acute
ASF,
which
include
localized
areas
of
cyanosis
in
the
skin,
an
enlarged,
friable,
dark-red
or
black
spleen,
and
enlarged,
reddened
gastrohepatic
and
renal
lymph
nodes
[3].
In
tissues
from
these
pigs,
viral
antigens
have
been
observed
by
direct
immunofluorescence
throughout
the
course
of
the
disease,
and
there
is
no
detectable
circulating
anti-ASFV
antibody.
In
contrast,
in
pigs
that
died
after
12
DPI
from
a
lesser
virulent
ASFV
infection,
such
as
ASFV
Dominican
Republic
1978
(DR-II)
or
Haiti
isolate,
the
spleen
was
still
enlarged
but
had
a
more
normal
color
and
was
less
friable;
in
addition,
tissue
sections
were
usually
negative
for
ASFV
antigen
by
direct
immunofluorescence,
and
serum
exhibited
circulating
anti-ASFV
antibodies
[4].
Anti-viral
antibodies
appear
in
the
serum
about
7
DPI
but
coexist
with
circulating
virus
[3].
It
is
not
known
why
antibody
does
not
neutralize
the
virus.
Mechanisms
regulating
the
immune
response
of
pigs
infected
with
any
of
the
ASFV
isolates
are
not
well
understood
[5].
Major
histocompatibility
complex
(MHC)
antigens,
termed
swine
leukocyte
antigens
(SLA)
in
pigs,
have
been
shown
to
be
important
in
immune
responses
to
viruses
and
in
the
recognition
and
clearance
of
viral
particles.
One
mechanism
of
inducing
viral
tolerance
is
down
regulation
of
MHC
antigen
expression,
resulting
in
a
failure
of
T
cells
to
recognize
and
eliminate
infected
cells.
Decrease
in
the
expression
of
MHC
antigens
has
been
described
in
infections
with
many
viruses
including
Friend
leukemia
[6],
ectromelia
[7],
vesicular
stomatitis
[8],
vaccinia
[9]
and
adenovirus
[10].
The
aim
of
this
study
was
to
examine
changes
in
cell
subset
localization
and
in
MHC
and
viral
antigen
expression
in
splenic
tissue
of
pigs
at
different
times
after
inoculation
with
either
the
highly
virulent
L60
or
the
moderately
virulent
DR-II
isolate
of
ASFV
to
determine
whether
MHC
regulation
was
ocurring
in
ASF.
Material
and
methods
Virus
and
animals
The
two
ASFV
isolates
used
in
these
studies
were
the
highly
virulent
Lisbon
virus
isolated
in
Portugal
in
1960
(L60)
[11]
and
the
moderately
virulent
virus
isolate,
derived
from
viremic
pig
blood
from
the
Dominican
Republic
outbreak
in
1978
(DR-II)
[3].
Twenty
Yorkshire
pigs,
6
to
8
weeks
old,
were
experimentally
inoculated
intramuscularly
with
ASFV
(100
hemadsorption
units
per
pig).
Groups
of
control
(non-inoculated)
and
inoculated
pigs
were
anaesthesized,
exsanguinated,
and
spleens
excised
at
varying
days
post
inoculation
(DPI).
Tissues
were
prepared
from
groups
of
pigs
sacrificed
at
each
time
point,
i.e.,
one
non-inoculated,
one
L60
and
one
DR-II
inoculated
pig
in
each
group,
except
at
6
and
15
DPI
when
most
L60
inoculated
pigs
had
already
died
from
ASF.
At
the
most
ASFV
effect
on
splenic
antigen
expression
147
important
time
points,
3
and
4
DPI,
three
independent
sets
of
pigs
were
used
to
collect
tissues.
As
an
additional
control
one
pig
was
inoculated
with
hog
cholera
virus
and
killed
at
4
DPI.
Monoclonal
antibodies
Five
murine
monoclonal
antibodies
(mAb)
with
specificities
for
porcine
lymphocyte
subsets
and
MHC
antigens
were
used
as
reagents:
74-22-15
(IgG2a,
k),
74-12-4
(IgG2b,
k)
PT85a
(IgG2a),
TH16a
(IgG2a),
MSA4
(IgG2a)
and
MSA3
(IgG2a).
Of
these,
74-22-15
recognizes
a
230
kDa
antigen
on
the
surface
of
macrophages
and
granulocytes,
and
the
74-12-4
mAb
recognizes
a
55
kDa
antigen
on
the
surface
of
CD4
T
helper
cells
[12].
Monoclonal
antibodies
specific
for
class
I
and
class
II
MHC
antigens,
PT85a
and
TH16a
mAb
rec-
ognizing
SLA
class
I
and
class
II
(SLA-DQ)
respectively,
were
kindly
provided
by
Dr.
William
Davis,
Washington
State
University,
Pullman,
WA
[13,
14].
Dr.
Craig
Hammer-
berg,
University
of
Michigan,
Ann
Anbor,
MI,
kindly
supplied
mAb
MSA3
that
recognizes
class
II
(SLA-DR)
antigens
and
MSA4
that
recognizes
the
CD2
T
cell
antigen
[14,
15].
Normal
ascites,
medium,
or
mAb
to
bluetongue
virus,
were
used
as
controls.
Hematoxylin
and
eosin
(HE)
stain
Spleens
from
non-infected
and
infected
animals
were
preserved
in
paraformaldehyde-lysine-
m-periodate
(PLP)
fixative
[16].
Tissues
were
embedded
in
paraffin,
sectioned
at
4-6µm
and
stained
with
Meyer's
HE.
Tissue
preparation
and
antibody
staining
Excised
spleens
were
cut
into
pieces
of
approximately
0.5
x
0.5
cm,
embedded
in
OCT
(Tissue,
Tek
Miles,
Illinois),
frozen
by
immersion
in
an
isopentane/liquid
nitrogen
slush
for
1
to
2min
and
stored
at
70
°C.
Cryostat
sections
were
cut
at
4-6µm,
placed
on
glass
microscope
slides
and
stained
by
a
modification
of
the
procedure
of
Minguez
et
al.
[17].
After
each
step,
the
sections
were
either
given
three
washes
with
Tris
buffer
saline
(TBS),
or
after
incubation
with
substrate,
rinsed
with
distilled
water.
Splenic
sections
were
first
covered
with
PLP
fixative
for
10
min
at
room
temperature
in
a
humidified
chamber
[16].
Non-specific
antibody
binding
was
blocked
by
incubation
with
1%
normal
swine
serum
in
TBS
for
30
min.
For
primary
indirect
immunostaining
(macrophages,
SLA
and
T
cell
antigen),
sections
were
incubated
with
the
mAb
supernatant,
or
with
medium
or
normal
ascites
as
a
negative
control,
overnight
at
4°C,
followed
by
incubation
for
45
min
at
room
temperature
with
rabbit-anti-mouse
IgG
coupled
to
alkaline
phosphatase
(Dako
Co.,
Copenhagen,
Denmark;
1
:
40
in
TBS).
The
substrate
solution
consisted
of
2
mg
naphthol
AS-MX,
dissolved
in
200
µI
of
N'N'
dimethylformamide
and
9.8
ml
of
0.1
M
Tris-buffer
pH
8.2
with
1
mg/ml
of
fast
blue
BB
(Sigma,
St.
Louis,
MO).
Levamisole,
(Sigma)
(2.4
mg/10
ml)
was
added
to
block
endogenous
alkaline
phosphatase
activity.
The
reaction
was
stopped
after
25
min
by
rinsing
with
distilled
water.
Secondary
indirect
immunostaining
of
ASF
viral
antigens
was
performed
by
incubating
sections
with
biotinylated
porcine
anti-ASFV
IgG
in
TBS
for
40
min
at
room
temperature.
To
inhibit
endogenous
peroxidase
in
the
tissue,
sections
were
incubated
with
phenylhy-
drazine
(0.1%)
in
PBS
for
45
min
at
37
°C,
and
then
with
avidin-biotin
peroxidase
complex
(Vector
Labs,
Burlingame,
CA) for
1
h
at
room
temperature.
The
substrate
solution
con-
sisted
of
4.5
ml
acetate
buffer
(0.1
M,
pH
5.2),
300
IA
stock
solution
(30
mg
of
3-amino-9-
carbazole
in
7.5
ml
of
N'N'
dimethylformamide)
and
60µl
hydrogen
peroxide
(3%).
This
solution
was
applied
to
the
slide
and
incubated
for
20
min
at
37
°C.
The
reaction
was
stopped
by
rinsing
with
distilled
water.
Slides
were
coverslipped
using
glycerol-gelatin
GG
148
M.
Gonzalez-Juarrero
et
al.
(Sigma)
as
a
mounting
medium.
Slides
were
reviewed
by
three
independent
scientists,
two
of
whom
read
the
slides
in
a
blinded
fashion.
Despite
differences
in
staining
intensity
due
to
variation
between
experiments,
the
same
patterns
of
staining
were
found
when
tissue
from
a
different
group
of
pigs
was
examined.
Results
Clinical
disease
and
histology
Animals
inoculated
with
either
ASFV
L60
or
DR-II
isolates
exhibited
typical
ASF
disease
signs.
Cells
in
the
macrophage
sheathed
arteries
(MSA)
in
H
&
E
stained
sections
of
spleens
from
ASFV
L60
inoculated
pigs
at
3
DPI
had
enlarged
nuclei
and
a
more
abundant
granular
cytoplasm,
when
compared
to
splenic
sections
from
non-infected
pigs
(data
not
shown).
In
the
periarterial
lymphoid
sheaths
(PALS),
lymphocyte
nuclei
were
enlarged
and
there
were
a
few
scattered
"holes"
containing
pyknotic
nuclei.
The
red
pulp
was
depleted
of
lymphocytes;
the
nuclei
of
the
reticular
cells
were
enlarged
and
some
cells
had
abundant
cytoplasm.
In
HE
stained
splenic
sections
from
an
ASFV
DR-II
inoculated
pig,
cells
in
the
MSA
had
apparently
normal
nuclei
and
abundant
granular
cyto-
plasm.
In
the
PALS,
lymphocyte
nuclei
were
hyperchromatic,
round
and
densely
packed,
with
scattered
mitotic
figures.
ASFV
antigen
expression
Viral
antigen
expression
was
found
to
be
maximum
between
3-4
DPI
for
both
ASF
virus
isolates.
ASFV
antigen
was
localized
in the
red
pulp,
in
the
marginal
zone
of
the
PALS
and
in
occasional
cells
in
the
lymphoid
follicles.
Staining
for
viral
antigens
was
not
seen
in
sections
from
the
noninfected
pig
(Fig.
1
A).
In
splenic
sections
from
DR-II
infected
pigs,
ASFV
antigen
was
present
in
distinct
cells
in
the
red
pulp
and
in
cells
at
the
periphery
of
the
MSA
(Fig.
1
C),
while
in
spleens
from
ASFV
L60
infected
pigs,
the
ASFV
antigen
was
concentrated
in
the
red
pulp
and
it
was
not
possible
to
distinguish
between
infected
cells
in
the
MSA
and
cells
in
the
red
pulp
(Fig.
1
B).
Macrophage
localization
Macrophage
distribution
in
splenic
tissue
from
ASFV
infected
pigs
was
im-
munohistologically
determined
using
mAb
74-22-15.
In
non-infected
pigs,
mac-
rophage
antigen
was
expressed
in
the
red
pulp
but
not
in
the
white
pulp
(Fig.
1
A).
Positive
staining
in
the
red
pulp
had
two
appearances:
one
was
an
intense
membrane
staining
of
many
individual
cells
and
the
other
was
a
weak
staining
throughout
the
MSA.
Spleens
from
pigs
at
2
DPI
with
ASFV
L60
had
the
same
macrophage
distribution
as
the
non-infected
spleens,
except
that
there
was
a
slight
increase
in
staining
of
the
MSA,
and
more
numerous
stained
cells
in
the
red
pulp.
Very
few
cells
contained
ASFV
antigen.
At
3
DPI
with
ASFV
L60,
there
appeared
to
be
a
general
decrease
in
macrophage
labeling;
there
were
fewer
labeled
cells
in
the
red
pulp
and
no
labeling
of
the
MSA
(Fig.
1
B).
In
V3
7,
t
••'
1
A
e
:
r
ASFV
effect
on
splenic
antigen
expression
149
••••
1
B
2A
.1
.
I
I
.
.
"••
...It
.•
=
-
.
.
2B
1
-
1t•
-4.
1E
7
.4
Fig.
1.
Dual
antibody
staining
of
cryostat
spleen
tissue
sections,
stained
with
74-22-15,
anti-
macrophage
mAb
(A—C)
and
with
alkaline
phosphatase
coupled
anti-mouse
Ig
and
substrate
(blue).
All
sections
were
then
stained
with
biotinylated
porcine
IgG
anti-ASFV
followed
by
peroxidase
coupled
avidin
(red).
Tissue
from
non-infected
pigs
(A),
from
ASFV
L60
inoculated
pigs
at
3
DPI
(B)
and
from
ASFV
DR-II
inoculated
pigs
at
3
DPI
(C)
Fig.
2.
Dual
antibody
staining
of
cryostat
spleen
tissue
sections
from
non-infected
control
pigs
(A),
from
pigs
inoculated
with
ASFV
L60
isolate
(B)
or
from
ASFV
DR-II
infected
pigs
(C)
at
4
DPI.
Spleen
sections
were
stained
with
anti-SLA
class
II
mAb,
MSA3
(A—
C),
and
anti-mouse
IgG
coupled
to
alkaline
phosphatase
(blue)
and
then
with
porcine
IgG
against
ASFV
antigen
coupled
to
biotin
(red)
150
M.
Gonzalez-Juarrero
et
al.
the
red
pulp
it
was
possible
to
find
cells
labeled
for
ASFV
antigen
with
and
without
immunostaining
for
the
74-22-15
antigen.
At
4
and
6
DPI,
the
decrease
in
the
number
of
cells
with
this
macrophage
antigen
was
even
greater.
Spleens
from
3
and
4
DPI
with
the
ASFV
DR-II
isolate
(Fig.
1
C)
had
either
no
or
minor
decrease
in
labeling
for
macrophage
antigen
in
the
red
pulp
but
had
a
more
intense
labeling
of
the
MSA
(Fig.
1
C).
In
spleens
from
pigs
at
15
DPI
with
the
DR-II
isolate,
the
intensity
and
distribution
of
the
macrophage
antigen
had
returned
to
the
level
of
spleens
from
non-infected
pigs.
Spleens
from
the
pig
infected
with
hog
cholera
virus
had
a
generalized
more
intense
labeling
for
macrophage
antigen
at
4
DPI
than
did
spleens
from
non-infected
pigs
(data
not
shown).
T
cell
localization
Splenic
sections
from
non-infected
pigs
incubated
with
the
mAb
74-12-4
that
recognizes
CD4
+
T
helper
cells
had
cells
labeled
with
moderate
intensity
around
the
central
artery
in
the
PALS
and
red
pulp.
Splenic
sections
from
DR-II
infected
pigs
had
an
increased
number
of
CD4
+
cells
in
the
PALS
and
red
pulp.
Sections
from
L60
infected
pig
spleens
at
3
DPI
had
a
similar
appearance.
Use
of
an
anti-CD2
mAb
(MSA4)
that
recognizes
all
peripheral
T
cells,
showed
more
cells
labeled
in
the
sections
and
the
same
pattern,
of
increased
numbers
of
positive
cells,
in
the
PALS
and
red
pulp
(data
not
shown).
SLA
class
I
expression
Splenic
sections
from
non-infected
pigs
labeled
for
SLA
class
I
had
only
slight
differences
in
intensity
of
label
between
the
different
areas;
the
germinal
centers
and
marginal
zones
of
the
PALS
were
more
intensely
labeled
than
the
mantle.
The
MSA
had
a
less
intense
label
than
the
rest
of
the
red
pulp
(Table
1).
Splenic
section
from
pigs
at
2
DPI
with
the
ASFV
L60
isolate
had
a
slight
decrease
in
SLA
class
I
expression
when
compared
to
non-infected
controls.
At
3
DPI,
there
was
a
very
pronounced
decrease
in
SLA
class
I
in
the
marginal
zone
of
the
PALS
and
in
the
red
pulp;
intensity
of
label
in
the
germinal
centers
was
similar
to
that
in
non-infected
pigs.
Splenic
sections
from
pigs
inoculated
with
ASFV
DR-II
isolate
showed
similar
loss
of
SLA
class
I
expression
at
3
DPI.
At
4
DPI,
however,
there
was
a
marked
difference
in
the
expression
of
SLA
class
I
in
splenic
sections
of
ASFV
L60
and
DR-II
infected
pigs.
Sections
from
ASFV
L60
infected
pigs
had
a
pronounced
decrease
in
SLA
class
I
in
the
marginal
zone
of
the
PALS
and
in
the
red
pulp;
germinal
center
labeling
was
similar
to
the
non-infected
pigs.
In
contrast,
sections
from
ASFV
DR-II
infected
pigs
had
an
increase
in
the
intensity
of
label
for
SLA
class
I
in
the
PALS
and
a
mild
increase
in
the
labeling
of
the
MSA.
Examination
at
higher
magnification
revealed
that
many
cells
expressed
both
ASFV
antigens
and
normal
levels
of
SLA
class
I,
however,
there
were
also
many
cells
that
expressed
ASFV
antigens
but
either
no
or
very
low
SLA
class
I.
At
6
DPI
splenic
sections
from
pigs
ASFV
effect
on
splenic
antigen
expression
151
Table
1.
Comparison
of
antigen
expression
on
ASFV
infected
spleen
sections
DPI
Antigen
White
pulp
Macrophage
sheath
area
Red
pulp
NI
L60
DR-II
NI
L60
DR-II
NI
L60
DR-II
2
Macrophage
+
+
ND
+
+
+
+
ND
+
+
+
+ +
ND
SLA
class
I
+
+
+ +
ND
+
+
ND
+
+
+
+
+
ND
SLA
class
II
+
+ + +
ND
ND
+
+
+ +
ND
ASFV
ND
/
+
ND
/
+
ND
3
Macrophage
+
/
+ +
x
+ + + +
+
+ +
+
SLA
class
I
+
+
+ +
+
+
+ +
+
+
+
SLA
class
II
++
++
+++
++
+
+
ASFV
++
++
+++
+++
4
Macrophage
+/—
++
x
+++
++
+/—
+
SLA
class
I
+
+
+
+
+
+
+
+ +
+
SLA
class
II
++
++
+++
+++
++
+++
ASFV
++
++
+++
+++
6
Macrophage
ND
+
+
ND
+
+
ND
SLA
class
I
+ +
+
+
+
ND
+
ND
+
+
ND
SLA
class
II
+ + + +
+
ND
+
/
ND
+
ND
ASFV
+ +
+
+
15
Macrophage
ND
+
ND
+
+ +
+
ND
+
+
SLA
class
I
+
ND
+
+
ND
+
+ +
ND
+ +
SLA
class
II
ND
+
+
ND
+ +
ND
+
+
ASFV
ND
ND
ND
Summary
of
results
of
double
antibody
staining
for
ASFV
antigens
and
for
macro-
phages,
SLA
class
I
or
SLA
class
II
on
frozen
splenic
sections
from
non-infected
(NI)
or
pigs
inoculated
with
Lisbon
60
(L60)
or
Dominican
Republic
II
(DR-II)
ASFV
isolates.
Scoring
of
the
staining
was
as
follows:
no,
+
minimal,
+ +
extensive,
and
+
+ +
very
pronounced
staining
for
mAb,
x
area
could
not
be
identified
in
these
tissues
ND
Not
done
inoculated
with
the
ASFV
L60
isolate
still
had
a
marked
decrease
in
SLA
class
I
in
the
red
pulp,
but
a
slight
increase
in
labeling
in
the
germinal
centers
and
MSA.
At
15
DPI,
splenic
sections
from
pigs
inoculated
with
the
ASFV
DR-II
isolate
had
a
pattern
and
intensity
of
labeling
for
SLA
class
I
similar
to
the
non-infected
pigs
(Table
1).
In
hog
cholera
infected
spleen
(4
DPI),
the
expression
of
SLA
class
I
was
higher
than
in
control
tissue
(data
not
shown).
Expression
of
class
I
antigen
appeared
in
the
red
pulp;
in
the
PALS
there
was
almost
no
staining.
SLA
class
II
expression
Splenic
SLA
class
II
were
detected
using
mAb
MSA3
(SLA-DR
specific)
and
TH16a
(SLA-DQ
specific).
In
non-infected
splenic
sections,
TH16a
labeled
more
152
M.
Gonzalez-Juarrero
et
al.
cells
than
did
MSA3
(Fig.
2A).
Both
mAb
labeled
a
high
percentage
of
cells
in
the
PALS;
the
label
was
more
homogeneous
and
weaker
in
the
marginal
zone
than
in
the
germinal
centers.
Scattered
cells
in the
red
pulp
were
labeled
more
intensely
than
any
cell
in
the
PALS.
Cells
in
the
MSA
were
not
labeled
(Fig.
2
A).
Splenic
sections
from
pigs
inoculated
with
the
ASFV
L60
isolate
had
a
slight
decrease
in
expression
of
SLA
class
II
at
2
DPI.
At
3
DPI
(Fig.
2
B),
PALS
marginal
zones
were
negative
for
SLA
class
II
but
the
red
pulp
still
had
a
considerable
number
of
intensely
labeled
cells.
At
4
DPI
there
were
fewer
cells
labeled
in
the
red
pulp
than
at
3
DPI.
At
6
DPI,
there
were
no
labeled
cells
in
infected
areas
of
the
spleen,
but
in
non-infected
areas
(lymphoid
tissue)
there
was
an
increase
in
SLA
class
II
label
(data
not
shown).
Splenic
sections
from
pigs
at
3
DPI
with
the
ASFV
DR-II
isolate
had
de-
creased
SLA
class
II
expression
similar
to
the
ASFV
L60
infected
pigs
at
3
DPI.
At
4
DPI
(Fig.
2
C),
the
staining
in
lymphoid
areas
was
more
intense
in
spleens
from
DR-II
infected
than
non-infected
pigs;
there
was
more
SLA
class
II
expression
in
areas
of
the
MSA,
which
were
not
stained
in
controls.
This
staining
was
also
more
intense
with
the
mAb
MSA3
than
with
TH16a.
Analyses
at
higher
magnification
indicated
that
cells
that
express
high
levels
of
ASFV
antigens
coexpress
either
no
or
high
levels
of
SLA
class
II.
Splenic
sections
from
hog
cholera
infected
pigs
had
a
more
intense
label
for
SLA
class
II
than
did
sections
from
non-infected
pigs
and
there
was
no
differential
staining
in
the
MSA
as
had
been
seen
in
ASFV
infected
spleens.
Discussion
These
studies,
summarized
in
Table
1,
demonstrated
that
there
are major
changes
in
macrophage
and
T
cell
numbers
and
in
the
expression
of
SLA
and
viral
antigens
of
spleens
from
pigs
at
varying
intervals
after
inoculation
with
ASFV
isolates
of
different
virulence.
The
first
major
difference
was
observed
in
HE
sections,
where
L60
infection
caused
more
severe
histologic
changes
than
did
the
DR-II
isolate.
In
the
L60
infected
spleens
early
in
infection,
there
was
lymphoid
depletion
plus
red
pulp
necrosis
which
later
was
evident
in
the
white
pulp.
In
contrast,
in the
DR-II
infected
spleens,
not
only
were
these
changes
minimal,
but
there
were
actually
mitotic
figures
in
the
lymphoid
tissues
indi-
cating
replication.
The
results
of
labeling
splenic
sections
with
mAb
74-22-15,
which
specifically
recognizes
macrophages
and
granulocytes,
correlate
with
the
findings
in
the
HE
stained
sections.
In
splenic
sections
from
pigs
inoculated
with
ASFV
L60,
there
was
an
inverse
correlation
between
macrophage
staining
and
virus
antigen
expression.
These
changes
showed
that
the
macrophage
population
responded
to
the
L60
virus
infection
as
soon
as
2
DPI
with
a
dramatic
decrease
in
mac-
rophage
staining
at
times
of
high
viral
antigen
expression.
With
DR-II
infection,
viral
staining
was
little
changed
from
3-4
DPI,
although
there
was
an
increase
in
staining
for
macrophages
in
the
MSA.
Taken
together,
the
HE,
macrophage,
ASFV
effect
on
splenic
antigen
expression
153
and
viral
staining
data
indicate
that
as
the
duration
of
infection
lengthened
there
was
increasing
necrosis
and
decreasing
numbers
of
macrophages in
the
L60
infected
spleens,
whereas
there
was
either
no
change
in
some
areas,
or
cellular
proliferation
and
increased
numbers
of
macrophages
in
other
areas,
of
the
DR-II
infected
spleens.
The
increase
in
macrophage
staining
could
be
in-
terpreted
as
an
increased
number
of
macrophages,
possibly
due
to
recruitment
from
the
circulation;
as
a
change
in
the
macrophage
population
due
to
ma-
turation
of
resident
cells;
or
both.
The
first
interpretation
correlates
well
with
the
increase
in
blood
monocyte
numbers
at
4
DPI
in
pigs
infected
with
ASFV
DR-II
[18],
whereas
the
second
is
supported
by
the
fact
that
MSA
macrophages
that
were
negative
for
class
II
SLA
in
spleen
sections
from
control
pigs
became
class
II
SLA
positive
at
4
DPI
of
ASFV
DR-II
infection.
Since
macrophages
are
required
as
accessory
cells
for
antigen
and
lectin
induced
mitogenesis
and
other
immunological
functions
[19],
this
viral
destruction
of
macrophages
could
explain
why,
unlike
moribund
animals,
cells
from
ASF
recovered
pigs
have
a
normal
response
to
T
cell
lectins
in
vitro
[20].
In
fact,
our
data
with
either
anti-CD4
or
anti-CD2
mAb,
as
well
as
earlier
studies
by
Minguez
et
al.
[17],
show
that
there
is
a
major
increase
in
T
cells
in
the
spleen
by
3
to
4
DPI
with
ASFV.
Pig
lymphocyte
homing
data,
reviewed
recently
by
Pabst
and
Binns
[21],
clearly
prove
that
lectin
and/or
antigen
stimulation
will
signal
alterations
in
cell
movement
and,
subsequently,
in
cell
function
so
that
defense
mechanisms
can
quickly
be
operative
at
local
areas.
Expression
of
SLA
class
I
and
class
II
in
the
spleen
was
decreased
at
3
DPI
in
both
ASFV
L60
and
DR-II
infections,
possibly
due
to
suppression
of
synthesis
of
SLA
during
the
height
of
ASFV
replication
or
to
changes
in
cell
subpop-
ulations.
At
4
DPI,
splenic
sections
from
L60
inoculated
pigs
continued
to
exhibit
decreased
SLA
expression
and,
at
6
DPI,
a
slight
reversal
in
SLA
expression
was
apparent,
however,
severe
splenic
necrosis
was
also
observed.
In
contrast,
at
4
DPI,
the
decrease
in
SLA
expression
in
spleens
from
the
ASFV
DR-II
infected
pigs
had
not
only
been
reversed
but
the
expression
of
class
I
and
II
SLA
in
these
animals
was
even
more
intense
than
in
the
controls,
suggesting
activation
of
cells
in
the
infected
tissue.
In
addition,
the
MSA,
which
were
negative
in
spleens
from
uninfected
pigs,
became
positive
for
SLA
class
II
expression
in
spleens
from
pigs
infected
with
ASFV
DR-II
or
with
hog
cholera
virus.
These
results
correlate
with
the
observations
mentioned
above
that
pigs
infected
with
ASFV
DR-II
seem
to
initiate
an
early
and
normal
immune
response
whereas,
in
ASFV
L60
infected
pigs,
this
is
delayed
or
non-existent.
The
increase
in
SLA
class
I
and
II
expression
at
4
DPI
may
be
due
to
the
release
of
soluble
factors
such
as
interferon
gamma,
which
has
been
described
as
a
potent
stim-
ulator
of
MHC
class
I
and
class
II
antigen
expression
[22].
This
view
is
supported
by
the
fact
that
spleen
sections
from
pigs
infected
with
hog
cholera,
a
virus
which
is
known
to
stimulate
high
interferon
production
[23],
had
higher
expres-
sion
of
SLA
than
controls.
Whether
the
increase
in
SLA
expression
seen
in
spleens
from
ASFV
DR-II
infected
pigs
is
due
to
gamma
interferon
or
to
other
154
M.
Gonzalez-Juarrero
et
al.
soluble
factors
remains
to
be
deter
mined.
It
is
known
that
porcine
recombinant
gamma
interferon
inhibits
ASFV
replication
in
vitro
[24].
Studies
on
the
in-
duction
of
gamma
interferon
during
early
ASFV
infection
are
currently
being
pursued.
It
also
would
be
interesting
to
analyze
the
production
of
other
soluble
factors
such
as
interleukin-3
and
granulocyte-macrophage-colony
stimulatory
factor
during
the
infection
since
Genovesi
et
al.
[25]
have
been
able
to
induce
swine
peripheral
blood
monocyte
proliferation
in
culture
by
using
macrophage-
colony
stimulatory
factor.
Presence
of
this
factor
in
the
infected
tissue
could
also
explain
why
ASFV
DR-TI
infected
pigs
have
attracted
an
increased
number
of
macrophages
to
the
splenic
MSA.
Some
viruses,
such
as
lactic
dehydrogenase
virus
[26],
Semliki
Forest
virus
[27]
and
cytomegalovirus
[28]
are
reported
to
use
MHC
antigens
as
viral
receptors.
In
the
analysis
of
individual
cells,
we
observed
infected
cells
coex-
pressing
viral
antigen
and
SLA
class
II;
we
also
observed
cells
which
expressed
only
viral
antigens,
thus
indicating
that
class
II
SLA
expression
is
not
necessary
for
ASFV
infection.
These
data
were
confirmed
by
our
in
vitro
studies
in
which
enriched
pig
monocytes
were
infected
with
ASFV
and
in
which
both
class
II
positive
and
negative
pig
monocytes
were
equally
likely
to
be
infected
with
ASFV.
Moreover,
treatment
of
these
monocytes
before
infection
with
anti-SLA
class
II
mAb
caused
no
decrease
in
number
of
ASFV
infected
cells
(M.
Gonzalez
Juarrero
et
al.,
manuscript
in
prep.).
In
summary,
we
found
extensive
necrosis,
decreased
density
of
macrophage
staining,
and
decreased
expression
of
MHC
antigens
in
spleens
from
ASFV
L60
infected
pigs
as
compared
to
proliferation
of
the
lymphoid
tissue
and
increased
density
of
macrophage
staining
and
MHC
expression
in
spleens
from
ASFV
DR-II
infected
pigs.
These
changes
in
infected
cells
and
in
expression
of
MHC
antigens
in
pigs
surviving
ASFV
infection
correlate
well
with
the
virulence
of
the
virus
isolate
and
may
explain
the
differences
reported
previously
in
the
immune
response
of
animals
infected
with
highly
virulent
versus
mod-
erately
virulent
ASF
viruses.
Acknowledgements
The
authors
thank
Ms.
M.
Diaz
for
her
technical
assistance
and
Mr.
S.
E.
Perlman
for
his
library
assistance.
The
authors
are
also
grateful
to
Dr.
William
Davis,
Washington
State
University,
and
Dr.
Craig
Hammerberg,
University
of
Michigan,
for
their
generous
gifts
of
hybridoma
cell
lines
and
to
Drs.
C.
Brown,
G.
Genovesi,
W.
Laegreid,
and
W.
Davis
for
their
critical
reviews
of
the
manuscript.
This
work
was
supported
by
the
US-Spain
Joint
Committee
for
Scientific
and
Technological
Cooperation,
program
#
G11.
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Authors'
address:
Dr.
J.
Lunney,
Helminthic
Diseases
Laboratory,
LPSI,
ARS,
B
1040,
Beltsville,
MD
20705,
U.S.A.
Received
May
1,
1991