have shown that horseradish peroxidase. (HRP) can cause murine thioglycollate-induced peritoneal macrophages. (M#{248})to produce both tumor necrosis ...
Journal
of Leukocyte
Biology
50:61 5-623
(1991)
Induction of Tumor Necrosis Factor and MacrophageMediated Cytotoxicity by Horseradish Peroxidase and Other Glycosylated Proteins: The Role of Enzymatic Activity and LPS Doris Department
L. Lefkowitz,
of Biological
Sciences,
Kevin
Texas
Mills,
Aaron
Recent studies by these investigators cause
murine
Castro,
and
Tech University (D.L.L., KM., AC.), Health Sciences Center (S.S.L.),
Stanley
and Department Lubbock
have shown that horseradish
thioglycollate-induced
peritoneal
macrophages
S. Lefkowitz
of Medical
peroxidase
Microbiology,
Texas
Tech
(HRP) can
(M#{248})to produce
both
tumor
necrosis factor (TNF) and enhance macrophage-mediated cytotoxicity (MMC) to 3T1 2 target cells. The present study identifies the roles of both enzymatic activity and contaminating lipopolysacharides (LPS) (1 ng) on these activities. The addition of 100 ng/ml of polymyxin B (PB) to enzymatically active HRP significantly reduced TNF production but did not affect MMC. Enzymatically inactive HRP (DHRP) was more effective than HRP in both TNF production and MMC but was not affected by PB. The inability of PB to modify DHRP-induced TNF suggests that LPS was not required. The induction of TNF and MMC in the absence of LPS was also corroborated by similar studies using M#{248} from endotoxin-resistant C3H/HeJ mice. Glycosylated proteins such as HRP, DHRP, and mannosylated
bovine
(mannosyl-fucosyl behaved suggest
serum
receptor
albumin
(M-BSA)
are known
[MFRJ) on the surface
to bind to mannose
of MO. In the present
similarly to DHRP in that it induced both TNF secretion and MMC. that binding to the MFR may be sufficient to induce TNF secretion
addition,
the data suggest
DHRP-induced
that neither
enzymatic
activity
receptors
studies,
M-BSA
These results and MMC. In
nor LPS was required
for
TNF.
Key words:
3T12 target
cells, C3H/HeJ
mice, thioglycollate-induced
peritoneal
macro-
phages
INTRODUCTION
combination
Macrophages
(M#{248})play
a central
role
in the
initiation
of both cellular and humoral immunity I 1 ] . A number substances have been shown to activate M#{216}to
cytotoxic
state
in
popolysaccharide
and
vitro
(LPS)
a number
including from
interferon
gram
of carbohydrates
negative
[28]
such
(IL-l)
interleukin-l
(TNF)
[9].
TNF,
a
In addition
number
of
and
to the
y,
including
and fucoidan [16,17]. of immunoregulatory
as
tumor
maleyl
necrosis
have
li-
role
of the
secretory
ities and probably
M#{248} these cells defenses against
cancer
by
of activated the major
cytotoxicity
reports peroxidases
from can
Peroxidases are which catalyze
H2O2.
© 1991
It
is well
Wiley-Liss,
this laboratory [2 1 ,23] have function as immunomodula-
a group of the oxidation established
Inc.
that
heme-containing enof certain substrates these
enzymes,
been
in
TNF
and
became
by a target
cell
assay
were
undertaken
studies
activity
enzymatic induced Received
of
of peroxidases
HRP
was
requests:
Sciences,
P.O.
7. 1991
the
present
studies
Doris Box
4149,
;
L.
and
cytocidal to determine
required
exact
if
for secre-
role
show
of
that
Tech
for
LPS
in
neither
by
HRPM#{248}ex-
Department
of
Biological
University.
Lubbock,
February
Lefkowitz, Texas
in
[20,23].
LPS was required TNF production accepted
M#{248}exor
of MMC. In addition, the immunomodulatory
to define
The
activity nor MMC. However,
Reprint
79409.
was
reactions.
January
a potent
viruses,
that thioglycollate-induced lactoperoxidase, microperoxidase,
tion of TNF as well as induction central to an understanding of
these
form
peroxidase (HRP) free radicals [ 10] and cause hepatoma tumors in rats [ 1 1].
secreted
present
ion, bacteria,
Horseradish
to generate
reported HRP,
a halide
of killing
of Novikoff
as determined
enzymatic
and
[8,18].
shown
We have posed to
H207 capable
cells
the regression
effects
capac-
[1,2].
Previous shown that tors. zymes
functions one of
has
The
importance of TNF in macrophage-mediated (MMC) [9, 12] . Because of the various cytotoxic represent
mammalian
vitro
factor
described
triad”
myeloperoxidase
Stimulated substances
immunoregulatory
investigators
of the
organisms,
bovine serum albumin M#{248} are a major source
with
“cytotoxic
13, 1991.
TX
616
Lefkowitzetal.
posed reduced
to
a partial
enzymatically in the presence LPS
TNF Assay
was significantly B (PB) suggesting
TNF was assayed using murine L929 cells [20,23,26]. Each sample was replicated at least 3 times. This method is briefly described as follows: 100 pA of complete DMEM containing 4 x l0 cells were added to each well
requirement.
MATERIALS Mice
AND METHODS
Age-matched
male
and C3H/HeJ mice, SASCO (Omaha, Harbor,
active HRP of polymyxin
and
8-12 NE)
female
C57B1/6,
weeks old, or Jackson
of a 96 well tissue culture microtiter plate and incubated for 24 hr at 37#{176}C.The media were aspirated and 100 pA of fresh media were added to each well. One hundred pA of the samples to be tested were added to the first well of
CeH/HeN,
were obtained Laboratories
from (Bar
a column and serially diluted down the plate. After dilution of the samples, 100 pA of actinomycin D (1 jig/mI) were added to each well. The cells were incubated
ME).
Reagents
dium
periodate
were
purchased
from
Sigma
were prepared using a 0.22
jim
MA).
Millex-GS
filter
(Millipore,
Bedford,
twice 50%
DMEM. Phosphate-buffered as described previously
nm
(PBS) Certain
.
does
not
a 1 :25 dilution ml as assayed
Assay
cross
neutralizes on L929
for Endotoxin
react
with
lymphotoxin
greater cells.
than
250
[14]
and
U of TNF/0.
1
Collection
as described cervical 6-8 ml serum-free
previously dislocation, of PBS. DMEM
at a concentration microliters of cell a 96 well
tissue
each
well,
and
collected
mice
killed
of I x 106 cells/mI. suspension was added
culture
microliters
Briefly,
M#{248} were were
by
followed by peritoneal lavage with The cells were washed 3 times with and resuspended in complete DMEM
and incubated 2 hr removed by washing hundred
[22].
cluster
(Costar,
One hundred to each well of Cambridge,
the M#{216} were
neous where
DMEM
incubated
were 48 hr prior
to
to use.
50 pA of 0.033%
using
the
ical 50% point ing dilutions. TNF titer was
following
TNF
formula:
form y above
=
titers
were
cytotoxicity two
was simulta-
ax + b were solved and below the theoret-
Assay (MMC)
3Tl2
The
percent
and x = the reciprocal of the correspondThen 0.50 was substituted for y and the calculated and expressed as U/l00 p.1.
was
assayed
Briefly, M#{216} were above. After 48 hr
DMEM containing well. Control wells tor:target culture
hundred microliters of were used to lyse the was measured at 550
reader.
The
as described collected incubation.
the peroxidase received DMEM
cells
were
cell ratio supernatants
used
as
and
previcultured 100 p.1 of
were added to each without peroxidase.
target
cells
of 16: 1 . After were aspirated
6 hr and
at an
effec-
incubation, saved for
the TNF
One hundred microliters containing 6 x l0 3T12 target appropriate wells. Another 100 were added to each well and the another 42 hr. At this time the phosphate-buffered formalin for
of complete DMEM cells were added to the p.1 of complete DMEM cultures were incubated cells were fixed in 10% 10 mm, and stained with
0.5%
borate
assay.
methylene
blue
The plates were unbound stain, extracted
with
0. 1 N HCI,
at 660 nm using wells containing the
wells
% cytotoxicity
.
buffer
at pH
8.4.
buffer to remove dry. The dye was
absorbance
was
measured
plate reader. The O.D. of the substracted from the O.D. of
M#{248}plus
calculated
.
and
a microtiter M#{248} was
containing was
in 10 mM
washed with borate and allowed to air
3T12
cells
and
the
cyto-
as follows:
r
were Two
added
plate
follows.
equations of the y = % cytotoxicity
toxicity
MA)
at 37#{176}C.Non-adherent cells twice with 100 p.1 of DMEM. of complete
determined
NIH
peritoneal
as
Cytotoxicity
Activity
Thioglycollate-induced
a microtiter
ously [23,32]. as described
All solutions were tested for endotoxin activity using a Limulus amoebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA). Solutions containing 8.2 jiM HRP contained 1 .0 ng LPS. All other media and reagents used contained 0.5 ng of LPS/ml. MO
using
MO Cytotoxicity
DMEM preparations were supplemented with PB (Sigma) at a concentration of 100 ng/ml. Rabbit polyclonal anti-TNF was obtained from Dr. George Gifford. This antibody
200 pA of PBS. Two in 100 mM NaH,PO4 Absorbance of the solutions
calculated
Dulbec-
saline
[ 1 8]
with
with ethanol
cells.
co’s modification of Eagle’s minimal essential medium (DMEM) (Gibco, Long Island, NY) supplemented with 2% fetal bovine serum (FBS) (Hyclone, Logan, UT), 25 mM HEPES (Sigma), and 25 jig!ml gentamicin (Sigma) were used for cultivation of M#{216}.This will be referred to as complete was prepared
by staining
neutral red (w/v) in PBS. After this incubation, cells were incubated for an additional hour at 37#{176}C,and washed
Chemical
Co. (St. Louis, MO). Activator solutions immediately prior to use and filter sterilized
hr at 37#{176}C, followed
20
HRP type V 1 , inactivated horseradish peroxidase (DHRP) (90%. In the presence of PB, secreted TNF was not detected using 2.7 p.M HRP; however, TNF induction by DHRP was not affected by PB. The production of TNF in the absence of LPS was confirmed using the LPS insensitive mouse strain C3H/ HeJ. Inactive HRP induced 4 times the amount of TNF (16 U/0. 1 ml) than HRP (-4 U/0. 1 ml) in these mice (data not HeJ mice
shown). All were markedly
M#{248} from C57B1/6. Since TNF was
TNF titers using M#{248}from lower than those obtained
induced
by DHRP,
which
C3H/ using
is a glyco-
P value
90 ± 6.0 167 ± 12.7
0.001 0.001
tM
1
