reovirus, when used in combination with. 1,3-bis(chloroethyl)-1- nitrosourea. (BCNU) chemotherapy, mediates the rejec- tion of murine ascites tumors. Surviving.
Tumor
necrosis Anthony
factor-a
L. Farone,
Department
induction
Patricia
of Micro&iology,
G.M.
Miami
O’Brien,
University,
and
Oxford,
Abstract: We have reported previously that reovirus, when used in combination with 1,3-bis(chloroethyl)-1nitrosourea (BCNU) chemotherapy, mediates the rejection of murine ascites tumors. Surviving anhnals reject a challenge with the same, but not a different, tumor, which suggests that tumor-specific immunity is induced by the treatment regimen. The present study was designed to characterize the interaction between reovirus and niurine peritoneal macrophages, both in vitro and in vivo, to determine whether such a relationship may play a role in immune modulation resulting in tumor rejection. The results demonstrated that reovirus can efficiently infect peritoneal macrophages in vitro and stimulate the secretion of tumor necrosis factor-alpha (TNF-a). In vivo administration of reovirus, however, did not produce high levels of infection in peritoneal exudate cells, even though the cells were stimulated to express detectable levels of membrane TNF-a. These results suggested that infection is not necessary for TNF-a expression and this hypothesis was supported by the observation that this expression was also stimulated in vitro by UV-inactivated reovirus. These findings suggest that one mechanism for immune stimulation by reovirus may be through the induction of TNF-a.-Farone, #{163}L.,O’Brien, P.C.M., Cox, D.C. Tumor necrosis factor-a (TNF-a) induction by reovirus serotype 3.J. Leukoc. Biol. 53: 133-137; 1993. Key
Words:
tumor
peritoneal
necrosis
factor-a
Macrophages tion
are
of the
come the infection
important
interest to the
it may
not
the
highly
and
reactive
function
nature
as a typical
bevirus
of
host
the
during
attachment and entry response leading to the resulting in the recruitcells important in viral
inflammatory
mediator
stimulated
by
virus infection is the cytokine, tumor necrosis factoralpha (TNF-a) (1-3, 5, 7). TNF-a, which is predominantly secreted by macrophages, is also expressed on the cell surface
(8).
tributed
study ment fected
to
sourea therapy mented
TNF-a;
biological those
are its antitumor and stimulation, cells
We have UV-inactivated the
Numerous
of
effects particular
activity and
and
cytolytic
have
been
interest immune effects
to cell on
atthis
recruitvirus-in-
(9-13).
previously reovirus
chemotherapy
reported serotype agent
that either infectious 3 in combination
or with
1 ,3-bis(2-chloroethyl)-1-nitro-
(BCNU) is a potent of murine tumors genome
of
causes
by two a variety
human
immune stimulator in the (14). Reovirus has a segdouble-stranded RNA (dsRNA) en-
Journal
protein coats. of mammalian
disease
Even
though types,
cell
reovirus it rarely
(15).
In this study, we have demonstrated that murine peritoneal macrophages can be productively infected by reovirus serotype 3 and that reovirus treatment stimulates the production of TNF-a both in vitro and in vivo. These results suggest that some of the immunomodulatory effects mediated by reovirus may occur through TNF-a.
MATERIALS
AND METHODS
Mice Female lan
CD2F1
(Balb/c
Laboratories,
x DBA/2)
mice,
Indianapolis,
IN),
age
were
7-9 wk used
in
(Harall
ex-
periments.
Macrophage
Collection
Thioglycollate-stimulated previously
the
cells
dish
macrophages
described
were
(Falcon
(16).
allowed
For
to
Labware,
were
the
adhere
to
Oxnard,
collected
infectious
CA)
center
a polystyrene for
90
mm
as assay,
petri at 37#{176}Cin
complete medium [RPMI 1640, Sigma Chemical Co., St. Louis, MO; 10% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories, Inc., Logan, Utah); 100 penicillin Co.,
and
and Louis,
St.
cells with
were
a cell
100 .Lg/ml MO]. After
removed
scraper,
and washed
streptomycin, incubation,
the
adherent cells with complete
Sigma non-
were colmedium,
counted.
Virus was
Reovirus
virus
3
C. Cox
capsidated can infect
lected
regula-
have recently targets for
serotype
45056
U/ml Chemical
#{149} membrane
induction
(1-5) and as potential
virus infection. In particular, may stimulate an inflammatory induction of immunomodulators, ment of immune effector One
Donald Ohio
adherent
in
response
focus of (6). Due
clearance.
#{149} Mac-2
#{149}reovirus
immune
macrophage,
ha ge
macrap
by reovirus
previously ing to the
purified
UV-inactivation Virus ticles
infectivity
7.2)
0.9
mM
infected
mouse
L929
cells
as
accord-
of Reovirus was
suspended
(D-PBS;
0.49
from
(17). Virus was then titrated of Shaw and Cox (18).
described procedure
inactivated
by
in Dulbecco’s mM
MgC12,
CaC12, 0.14
at a concentration
2.68 mM
irradiating
phosphate mM NaCl,
KCI,
1.47
8.03
mM
of 5 x 1012
virus
buffered
particles/mI
mM
par-
saline K1-12PO4,
Na2HPO4,
with
pH
a ger-
Abbreviations: BCNU, I ,3-bis(2.chloroethyl)-1-nitrosourea; BRMA?, Biological Response Modifiers Program; D-PBS, Dulbecco’s phosphate buffered saline; dsRNA, double-stranded RNA; FBS, fetal bovine serum; MOl, multiplicity of infection; pfu, plaque forming units; TNF.ct, tumor necrosis factor-alpha. Reprint requests: Anthony L. Farone, Harvard School of Public Health, Respiratory Biology Program. Department of Environmental Health, Bldg. 1, Rm. 307, 665 Huntington Ave., Boston, MA 20115. Received
June
of Leukocyte
5, 1992;
Biology
accepted
September
Volume
4, 1992.
53,
February
1993
133
micidal
lamp,
l.LW/cm2) was
(254
for
determined
Virus
nm)
mm.
15
at The
using
the
a distance
of
effectiveness
reovirus
15 of
titration
cm
(1250
Infectious
protocol.
vivo,
tion
virus
as
was
injected
described
above,
4 h before
above.
and
the
Antibody
Cells
macrophage
were
percentage
of
washed
collectwice,
infected
cells
plated
was
deter-
mined.
Culture
Macrophages
were
for TNF-a plated
at
medium in 24-well culture for 90 mm at 37#{176}C.After cells were removed with medium containing 1 lipopolysaccharide (LPS; MO), reovirus at a MOI equivalent to a MOI of was added to the adherent 4 h at 37#{176}Cand cell-free lected.
Release
3 x 106
cells/ml
in
complete
plates and allowed to adhere incubation, the nonadherent D-PBS, and 1 ml of complete .ig/ml Escherichia coli 0111:84 Sigma Chemical Co., St. Louis, of 10, UV-inactivated reovirus 10, or complete medium alone cells. Cells were stimulated for culture supernatants were col-
sample
(U)-neutralized
Detection
Peritoneal exudate mice injected with thioglycollate alone. to 1 x I 02 cells/mI azide
(w/v),
gelatin suspensions
Statistical
alone.
After
glutaraldehyde 1 h and stained
134
Journal
incubation,
Chemical equivalent
noninfectious the cells
Biology
St.
Louis, of
St. Louis, MO) for blue (Fisher Scien-
Volume
53,
St.
anti-murine CA) and San Mac-2
Louis,
MO;
Detroit, ml
were
MI]. then
TNF-cx antibody streptavidin-phycoerythrin Jose, CA) followed
monoclonal
0.1%
Cell stained (Pharby
antibody
60
tissue
%
Mac-2,
in
then
of Mac-2
% (%
subtracting binding
the the
percent isotype
used
in
with
mTNF-a
cells
Mac-2,
the
of cells controls.
following
+
%
nonadjusted
equation:
=
mTNF-a
mTNF-a
exhibiting The
x
100%.
Mac-2)
Analysis
LPS,
reovirus, or medium were fixed with 2.5%
(Sigma Chemical Co., with 0.05% methylene
of Leukocyte
Co., amounts
Co.,
Laboratories,
culture supernatant (M3/38.2.8.HL.2 hybridoma; a generous gift of Dr. Meena Subramanyam, Tufts University, Boston, MA) and 1 p.g biotin-conjugated goat affinity purified F(ab’)2 anti-rat IgG, whole molecule (Cappel Research Products, Durham, NC), followed by 1 p.g fluorescein-conjugated streptavidin (Pierce Chemical Co., Rockford, IL). Isotype controls were used to set markers on the double negative population. Data from the cell populations were acquired and analyzed with a FACScan flow cytometer (Becton-Dickinson). The percentage of the total Mac-2 cell population that was expressing mTNF-a was calculated by first determining the percentage of cells expressing Mac-2 alone, or, both Mac-2 and
j.tg/ml
(Sigma
In vivo
106 cells/0.1
anti-mouse
were
D
Chemical
Difco
with 1 .tg biotinylated Mingen, San Diego, (SAPE; Becton-Dickinson,
values
received
TNF-a
containing
rat
(U))
cells were collected as above from thioglycollate followed by reovirus or Cells were washed and resuspended in azide D-PBS [A-PBS; 0.1% sodium
Sigma
(w/v),
Culture supernatants were assayed in triplicate for TNF-a activity immediately after collection using the L929 cell cytotoxicity method (19). Briefly, 5 x 10 L929 cells/well were incubated at 37#{176}Cwith culture supernatants or recombinant TNF-a [Biological Response Modifiers Program (BRMP) standard] serially diluted in MEM supplemented with 5% FBS, penicillin, and streptomycin in 96-well microtiter plates for 18 h in the presence of 1 Actinomycin
activity
(U)
of Membrane
by
MO). Control wells infectious reovirus,
sample
sample
TNF-a specific
TN F-a Assay
of TNF-a
% Neutralization=
p.1 of
Macrophage
neutralization
TNF-u cytotoxic activity was specifically neutralized with anti-TNF-a antibody (Genzyme Corporation, Boston, MA) by incubating culture supernatants with antibody dilutions for 1 h at 37#{176}C before bioassay. Percent neutralization was determined as follows:
Assay
The percentage of cells infected with reovirus in vitro and in vivo was determined as described by Duncan et al. (17). For macrophage infection in vitro, the amount of reovirus required for the appropriate multiplicity of infection (MOI) was added to thioglycollate-elicited macrophages in 0.5 ml of RPMI 11640 medium without serum and incubated at 37#{176}C for 1 h. After incubation, the cells were washed two times with 5 ml of complete medium. The cell suspensions were then serially diluted and 2 ml of complete medium containing 10k, 10, or 102 cells added, in duplicate, to confluent monolayers of L929 cells in 6 well plates (Falcon) and allowed to incubate for 5 h at 37#{176}C before removal of medium and addition of agar overlays as described for the reovirus titration. Plaques were counted and the percentage of infected cells was determined. For determination of percent infection
as
Pittsburgh, PA) for 30 mm. The dye was with 0.33 N HCI and the absorbance (570 nm) was measured with a Mini-Reader II EUSA reader (Dynatech Laboratories Inc., Alexandria, VA). The activity of the culture supernatants was then determined from the standard curve.
previously with thioglycollate were injected plaque forming units (pfu) of reovirus in 0.2 or DPBS alone 4 h before peritoneal cell col-
Center
Co.,
released
Injection
Mice injected i.p. with l0 ml of D-PBS lection.
in
tific
inactivation
February
The levels mTNF-a
of TNF-a results were
variance
using
(Graphpad
1993
the
Software,
detected analyzed InStat
San
in
the L929 by one-way
statistical
Diego,
CA).
analysis
bioassay analysis program
and of
RESULTS
TABLE
Infectious Reovirus
Center Assay In Vitro
of Macrophages
Treated
with
Treatment of macrophages in vitro with reovirus resulted in a productive infection; however, the efficiency of infection was MO! dependent. Data from three independent infectious center assays (Table 1) demonstrate that 96 ± 4% of the macrophages were infected by a MO! of 100. A lower efficiency of infection of 37 ± 6% was observed when the cells were treated with a MOl of 10.
2.
Stimulation
of TNF-cz
by Reovirus
Production TNF-ct
Treatmenta
(U/ml)’
LPS
1280±316
Reovirus UV-reovirus
1840 2386
Medium
3 ± 63
control
aThioglycollateelicited appropriate
± 289 ± 233
macrophages
stimulator
or
complete
were
(medium
assay of the cell-free supernatants bUnits (U) of activity were
for TNF-ct determined
standard
are means
curve.
Data
presented
cultured
medium
activity. from ± SD of
for
4
h
control)
with
before
rTNF-u (BRMP) two independent
experiments.
Infectious Reovirus
Center In Vivo
Assay
of Macrophages
Treated
with neutralizing
antibody,
suggesting
that
the
activity
was
due
to TNF-a.
Administration of 10 pfu of reovirus i.p. produced very low levels of infectivity in the peritoneal macrophages. Results combined from two independent experiments demonstrated that 0.4 ± 0.05% of the macrophages were infected in animals injected with reovirus 4 h before cell collection.
Stimulation
of TNF-cz Production
by Reovirus
In Vitro
To determine if macrophages were stimulated to produce TNF-a by reovirus treatment, TNF-ct activity in macrophage culture supernatants treated with reovirus or UVinactivated reovirus was compared to supernatants from cells treated with 1 p.g/ml LPS in a L929 cytotoxicity assay. UV-inactivated virus was used to assess whether infectious virus was necessary for induction of TNF-a. Results combined from two independent experiments (Table 2) indicate that infectious and noninfectious reovirus are both capable of inducing TNF-a at levels greater than those stimulated by LPS. None of the agents produced any detectable cytotoxic effects when added directly to the L929 bioassay. Noninfectious virus produced the highest amount of TNF-a activity followed by infectious virus and LPS. Both of the values for virustreated cells were significantly different from each other as well as from the LPS-stimulated activity.
In Vivo Induction of Membrane Macrophages by Reovirus
TN F-a on Peritoneal
To determine if reovirus stimulated the expression of TNF-a on the surface of thioglycollate-elicited peritoneal exudate cells in vivo, peritoneal macrophages were elicited with thioglycollate 4 days before i.p. injection of reovirus or D-PBS. Four hours after reovirus administration, peritoneal exudate cells were collected and stained with fluorescent-labeled anti-TNF-a and anti-Mac-2 antibodies. Results combined from two independent experiments demonstrated significant differences in cells treated with reovirus in which 27.5 ± 2.3% of the total Mac2* cell population expressed mTNF-a compared with 8.9 ± 1.3% expressed on the untreated cells.
100 90 80 70
Neutralization Antibody
of TNF-a
Activity
by Anti-TNF-a 0
60
0
Supernatants containing TNF-a activity from reovirus, UV-inactivated reovirus, or LPS-treated cells were incubated with anti-murine TNF-a antibody to determine if the cytotoxic activity exhibited in the bioassay was due to TNF-a. Results in Fig. 1 demonstrate that the cytotoxic activity in reovirus, UV-inactivated reovirus, and LPS stimulated supernatants can be completely abrogated by
N
50
0 5
40
z
30 20 10
TABLE
1.
Percent
of Macrophages
Infection
Reovirus
Reovirus
Treatment
after
Anti-TNF Percent
(MOI)a
100
96±4
bData
serotype presented
macrophages
3 at multiplicities are
means
±
were
of infection SD
of three
incubated
1. Neutralization TNF.a-containing lated with 1 tg/ml Fig.
for
1 h
of 10 or 100 phi/cell.
independent
(Dilution)
Infectionb
37±6
amioglycollate.elicited
iO_3
10_2
10’
10
reovirus
0-
with
In Vitro
experiments.
with
of TNF-a
supernatants LPS
(0),
activity from reovirus
by anti-murine TNF-a peritoneal macrophages (#{149}), or UV-inactivated
(V) were incubated with dilutions of anti-TNF-a 37#{176}Cbefore assaying for L929 cytotoxicity. The sentative of experiments that have been repeated
Farone
d
at.
TNF-a
induction
antibody. stimureovirus
antibody for one h at above data are repreat least three times.
by
reovirus
135
DISCUSSION Macrophages
are
ing
inflammatory
a potent
multifunctional
cells
capable
response
upon
of
initiat-
activation
by
various agents (6). One important mediator of this response produced by activated macrophages is TNF-cz (20, 21). TNF-a is a cytokine that appears to mediate a variety of immunomodulatory and physiological effects, as well as being directly cytotoxic to some tumor cells (912, 22). We have previously demonstrated that reovirus/BCNU treatment affects significant therapy of murine ascites tumors. Resistance of the surviving animals to challenge with the tumor from which they were
cured,
but
not
therapy regimen The purpose of
a different
tumor,
causes tumor-specific this study was to
suggests
that
this
immunity (14). to characterize
begin
during
tumor
Initial
using
of
the
infectious
of reovirus infection Results suggested
could
established
ent
a
with
MO!
could
and
essentially
of bind
the
100. and
These productively
virus/cell
center
efficiency in vitro. be
assays
interaction
was
to determine
the
136
approximately untreated
Journal
was
from
the
supported
Ohio
by
Board
a
Research
Challenge
of Regents.
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