Journal of Leukocyte Biology 50:61 5-623 (1991)
© 1991 Wiley-Liss, Inc.
Induction of Tumor Necrosis Factor and Macrophage-Mediated Cytotoxicity by Horseradish Peroxidase and
Other Glycosylated Proteins: The Role of Enzymatic Activityand LPS
Doris L. Lefkowitz, Kevin Mills, Aaron Castro, and Stanley S. LefkowitzDepartment of Biological Sciences, Texas Tech University (D.L.L., KM., AC.), and Department of Medical Microbiology, Texas Tech
Health Sciences Center (S.S.L.), Lubbock
Recent studies by these investigators have shown that horseradish peroxidase (HRP) cancause murine thioglycollate-induced peritoneal macrophages (M#{248})to produce bothtumor necrosis factor (TNF) and enhance macrophage-mediated cytotoxicity (MMC) to3T1 2 target cells. The present study identifies the roles of both enzymatic activity andcontaminating lipopolysacharides (LPS) (�1 ng) on these activities. The addition of 100ng/ml of polymyxin B (PB) to enzymatically active HRP significantly reduced TNFproduction but did not affect MMC. Enzymatically inactive HRP (DHRP) was more effectivethan HRP in both TNF production and MMC but was not affected by PB. The inability ofPB to modify DHRP-induced TNF suggests that LPS was not required. The induction ofTNF 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, andmannosylated bovine serum albumin (M-BSA) are known to bind to mannose receptors(mannosyl-fucosyl receptor [MFRJ) on the surface of MO. In the present studies, M-BSAbehaved similarly to DHRP in that it induced both TNF secretion and MMC. These resultssuggest that binding to the MFR may be sufficient to induce TNF secretion and MMC. Inaddition, the data suggest that neither enzymatic activity nor LPS was required forDHRP-induced TNF.
Key words: 3T12 target cells, C3H/HeJ mice, thioglycollate-induced peritoneal macro-phages
INTRODUCTION
Macrophages (M#{248})play a central role in the initiation
of both cellular and humoral immunity I 1 ] . A number of
substances have been shown to activate M#{216}to the
cytotoxic state in vitro including interferon �y, li-popolysaccharide (LPS) from gram negative organisms,
and a number of carbohydrates [28] including maleylbovine serum albumin and fucoidan [16,17]. Stimulated
M#{248}are a major source of immunoregulatory substances
such as interleukin-l (IL-l) and tumor necrosis factor
(TNF) [9]. In addition to the immunoregulatory role ofTNF, a number of investigators have described the
importance of TNF in macrophage-mediated cytotoxicity
(MMC) [9, 12] . Because of the various secretory capac-
ities and cytotoxic functions of activated M#{248}these cells
probably represent one of the major defenses against
cancer [1,2].Previous reports from this laboratory [2 1 ,23] have
shown that peroxidases can function as immunomodula-
tors. Peroxidases are a group of heme-containing en-
zymes which catalyze the oxidation of certain substrates
by H2O2. It is well established that these enzymes, in
combination with H207 and a halide ion, form a potent
“cytotoxic triad” capable of killing bacteria, viruses, and
mammalian cells [8,18]. Horseradish peroxidase (HRP)
has been shown to generate free radicals [ 10] and cause
the regression of Novikoff hepatoma tumors in rats [ 1 1].
We have reported that thioglycollate-induced M#{248}ex-
posed to HRP, lactoperoxidase, microperoxidase, or
myeloperoxidase secreted TNF and became cytocidal in
vitro as determined by a target cell assay [20,23].
The present studies were undertaken to determine if
enzymatic activity of peroxidases was required for secre-
tion of TNF as well as induction of MMC. In addition,
central to an understanding of the immunomodulatory
effects of HRP was to define the exact role of LPS in
these reactions. The present studies show that neitherenzymatic activity nor LPS was required for HRP-
induced MMC. However, TNF production by M#{248}ex-
Received January 7. 1991 ; accepted February 13, 1991.
Reprint requests: Doris L. Lefkowitz, Department of Biological
Sciences, P.O. Box 4149, Texas Tech University. Lubbock, TX
79409.
616 Lefkowitzetal.
posed to enzymatically active HRP was significantly
reduced in the presence of polymyxin B (PB) suggesting
a partial LPS requirement.
MATERIALS AND METHODSMice
Age-matched male and female C57B1/6, CeH/HeN,
and C3H/HeJ mice, 8-12 weeks old, were obtained from
SASCO (Omaha, NE) or Jackson Laboratories (Bar
Harbor, ME).
Reagents
HRP type V 1 , inactivated horseradish peroxidase
(DHRP) (<0.01% enzymatic activity), bovine serum
albumin (BSA) essentially globulin free, mannosylated
BSA (M-BSA), galactosylated BSA (G-BSA), and so-
dium periodate were purchased from Sigma Chemical
Co. (St. Louis, MO). Activator solutions were prepared
immediately prior to use and filter sterilized using a 0.22
jim Millex-GS filter (Millipore, Bedford, MA). Dulbec-
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 DMEM. Phosphate-buffered saline (PBS)
was prepared as described previously [ 1 8] . Certain
DMEM preparations were supplemented with PB (Sig-
ma) at a concentration of 100 ng/ml. Rabbit polyclonal
anti-TNF was obtained from Dr. George Gifford. This
antibody does not cross react with lymphotoxin [14] and
a 1 :25 dilution neutralizes greater than 250 U of TNF/0. 1
ml as assayed on L929 cells.
Assay for Endotoxin Activity
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 Collection
Thioglycollate-induced peritoneal M#{248}were collected
as described previously [22]. Briefly, mice were killed by
cervical dislocation, followed by peritoneal lavage with
6-8 ml of PBS. The cells were washed 3 times with
serum-free DMEM and resuspended in complete DMEM
at a concentration of I x 106 cells/mI. One hundred
microliters of cell suspension was added to each well of
a 96 well tissue culture cluster (Costar, Cambridge, MA)
and incubated 2 hr at 37#{176}C.Non-adherent cells were
removed by washing twice with 100 p.1 of DMEM. Two
hundred microliters of complete DMEM were added to
each well, and the M#{216}were incubated 48 hr prior to use.
TNF Assay
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
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
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
20 hr at 37#{176}C,followed by staining with 50 pA of 0.033%
neutral red (w/v) in PBS. After this incubation, cells were
incubated for an additional hour at 37#{176}C,and washed
twice with 200 pA of PBS. Two hundred microliters of
50% ethanol in 100 mM NaH,PO4 were used to lyse the
cells. Absorbance of the solutions was measured at 550
nm using a microtiter plate reader. The TNF titers were
calculated as follows. The percent cytotoxicity was
determined using the following formula: two simulta-
neous equations of the form y = ax + b were solved
where y = % cytotoxicity above and below the theoret-
ical 50% point and x = the reciprocal of the correspond-
ing dilutions. Then 0.50 was substituted for y and the
TNF titer was calculated and expressed as U/l00 p.1.
MO Cytotoxicity Assay
Cytotoxicity (MMC) was assayed as described previ-
ously [23,32]. Briefly, M#{216}were collected and cultured
as described above. After 48 hr incubation. 100 p.1 of
DMEM containing the peroxidase were added to each
well. Control wells received DMEM without peroxidase.
NIH 3Tl2 cells were used as target cells at an effec-
tor:target cell ratio of 16: 1 . After 6 hr incubation, the
culture supernatants were aspirated and saved for TNF
assay. One hundred microliters of complete DMEM
containing 6 x l0� 3T12 target cells were added to the
appropriate wells. Another 100 p.1 of complete DMEM
were added to each well and the cultures were incubated
another 42 hr. At this time the cells were fixed in 10%
phosphate-buffered formalin for 10 mm, and stained with
0.5% methylene blue in 10 mM borate buffer at pH 8.4.
The plates were washed with borate buffer to remove
unbound stain, and allowed to air dry. The dye was
extracted with 0. 1 N HCI, and absorbance was measured
at 660 nm using a microtiter plate reader. The O.D. of the
wells containing M#{248}was substracted from the O.D. of
the wells containing M#{248}plus 3T12 cells and the cyto-
toxicity was calculated as follows:
r /meansO.D.of. . I I treated cultures
% cytotoxicity I 1 -1 X 100.I I mean O.D. ofL \control cultures
Peroxidase-Induced MO-Mediated Cytotoxicity 617
Chemiluminescence Assay
The methods used have been described previously
[2 1] . Briefly, M#{216}were collected from the peritoneal
cavity and washed 3 times in Eagle’s minimum essential
media without phenol red containing 1% BSA. The cells
were adjusted to a concentration of 1 X 106 cells/ml and
100 p.1 were added to an 8 X 50 mm tube (Evergreen
Scientific, Los Angeles, CA). After a 30 mm incubation
at 37#{176}C,100 p.1 of the test solution or 100 p.1 of media
were added to each tube plus an additional 100 p.1 of
opsonized zymosan and 30 p.1 of luminol . The tubes were
placed in a luminometer (Turner, model 20e) and five
2-mm counts were recorded. The results were plotted as
time vs. counts (light emission). All treatments were
done in duplicate and each experiment was repeated at
least 3 times. Each value represents the mean of the 2
replicate cultures.
Statistical Analysis of Data
A Student t-test was used to determine the significance
of the effects of peroxidases on TNF production and
MMC. A one way analysis of variance (ANOVA) was
used to examine the dose response effects of peroxidases
on MMC. The data were arcsin � transformed prior to
analysis. A Student Newman-Kuel, a posteriori test, was
performed on the transformed means to determine sig-
nificant treatment level effects among the different
groups. Non-transformed means are illustrated in the
appropriate figures.
RESULTS
The production of TNF by M#{248}exposed to HRP has
been reported previously [23] . In order to determine the
necessity for enzymatic activity with respect to TNF
production, DHRP was compared with enzymatically
active HRP. The results shown in Table 1 indicate that
enzymatic activity was not necessary for TNF produc-
tion. Furthermore, on a molar basis, DHRP induced
higher titers of TNF than HRP. TNF production induced
by 8.2 p.M HRP was significantly reduced in the presence
of PB . It can be seen that the presence of 100 ng/ml of PB
during the 6 hr TNF induction period reduced TNF
production by >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 shown). All TNF titers using M#{248}from C3H/
HeJ mice were markedly lower than those obtained using
M#{248}from C57B1/6.
Since TNF was induced by DHRP, which is a glyco-
TABLE 1. The Effect of PB on TNF Production by M4Exposed to Either the Enzymatically Active HRP orEnzymatically inactive HRP�
TNF (U/0. 1 ml)
+ PB
Treatment (100 ng/ml) - PB P value
HRP8.2 �zM
Exp. I 8 ± 0#{149}1b 90 ± 6.0 �0.001Exp. 2 6 ± 0.3 167 ± 12.7 �0.001
2.7 �tM
Exp. 1 <2 34 ± 4.4 �0.00l
Exp. 2 <2 25 ± 6.9 �0.00lDHRP
8.2 j�M
Exp. 1 2 1 8 ± 44.8 207 ± 46.5 N.S.Exp. 2 664 ± 178 454 ± 106.0 N.S.
0.82 �M
Exp. I 10 ± 0.9 1 1 ± 1.5 N.S.Exp. 2 33 ± 35 63 ± 1.0 N.S.
alhioglycollate..induced M4 from C57BI/6 mice were exposed to
either HRP or DHRP for 6 hr. Control wells were exposed to equalvolumes of media. After 6 hr, the supernatants were transferred to
L929 cells and assayed for the presence ofTNF. Titers are expressedas U/0.l ml.�‘Titers are the mean of 3 replicate cultures ± S.E.M.
sylated protein, studies were undertaken to determine if
other glycosylated proteins such as M-BSA or G-BSA
could also induce this activity. Experiments were done to
compare TNF production by M#{248}exposed to either
M-BSA or DHRP. The data in Table 2 illustrate that
M-BSA was capable of inducing higher levels of TNF
than DHRP. M-BSA induced between 150 and 300 U of
TNF/0. 1 ml compared with DHRP which induced 25-50
U/0. 1 ml. G-BSA induced 0-10 U of TNF (data not
shown). Mannan or BSA alone was totally ineffective in
this capacity. It can also be seen that the presence of PB
did not affect TNF production by either DHRP or
M-BSA.
Studies were initiated to look at binding properties of
HRP, DHRP, and M-BSA using chemiluminescence. It
is reported in the literature that both HRP and glycosy-
lated-BSAs bind to the mannosyl-fucosyl receptor (MFR)
of M#{216}[15]. HRP but not DHRP, induced chemilumi-
nescence (Fig. 1). Chemiluminescence of M#{248}exposed to
0.04 p.M HRP was inhibited in a dose dependent manner
by both DHRP and mannose (Figs. 1 , 2). M-BSA did not
induce chemiluminescence nor did it inhibit HRP-in-
duced chemiluminescence (data not shown). In the
absence of M#{216},the values obtained either declined or
remained level during the 10 mm observation period.
Other studies were undertaken to investigate the effects
of radicals on peroxidase-induced TNF. Sodium period-
ate, a free radical generator, was added to M#{216}cultures
TABLE 2. The Induction of TNF by M4 Exposed to -�0- Control
-*-
-0-
-n-
- - .- -
HRP 0.04 p.M
HRPO.04 p.M +DHRPO.l p.MDHRP 0.2 p.M
HRPO.04 p.M + DHRPO.I nM
HRP 0.075 pM; without M#{248}
I-.zC
c)
40
20
0 6 8 10
TIME (MIN.)
12
-0-
-�-
Control
HRP 0.04 p.M
im� 0.04 p.M + Man 50 mM
HRPO.04p.M+ Man5mM
HRP 0.04 p.M + Man 0.5 mM
CID
z
C
2 4 6 8
Time (mm.)
10
mannan nor BSA alone was capable of inducing MMC
under these conditions (data not shown).
The role of LPS in the induction of MMC was also
addressed through the use of C3H/HeJ mice. Studies
using M#{248}from the endotoxin-resistant mice showed that
618 Lefkowitz et al.
Glycosylated Proteinsa
Treatment Conc./ml + PB - PB
Glycosylated proteinsM-BSA
Exp. 1 0.485 j.�M 273 ± 307b 165 ± 43.3Exp.2 0.485 MM 230±41.2 149± 11.2
DHRPExp. 1 0.825 MM 43 ± 8.0 26 ± 1.1Exp. 2 0.825 j.�M 27 ± 4.5 49 ± 4.2
ControlsBSA
Exp. 1 30 j.�M <2 <2Exp. 2 30 �M <2 <2
MannansExp. 1 57 �M <2 <2Exp. 2 57 �M <2 <2
LPS 100 ng10 ng
lng
<2<2<2
172 ± 11.5549 ± 12.70
<2..
aThioglycollateinduced M4 were exposed to either media, M-BSA,
enzymatically inactive HRP, BSA, mannans, or LPS in the presence orabsence of PB. After 6 hr incubation, the supernatants were collected,transferred to L929 cells, and assayed for TNF. Titers are expressed as
U/0.l r,�l.bValues are mean of 3 replicate cultures ± S.E.M.
during the induction of TNF. It can be seen in Figure 3a
that 1 mM concentration of sodium periodate enhanced
HRP-induced TNF secretion 3 fold (P � 0.001). Higher
concentrations of sodium periodate were inhibitory.
Similar studies done with DHRP showed no statistically
significant effect on TNF production (Fig. 3b). The high
concentration of 4 mM inhibited both HRP and DHRP-
induced TNF.
Induction of MMC by HRP has also been reported
previously [23,32]. Studies were undertaken to deter-
mine if DHRP could function similarly to enzymatically
active HRP and induce MMC. Figure 4 shows that DHRP
was also capable of inducing cytotoxicity. At a concen-
tration of 8.25 p.M. DHRP induced more cytotoxicity
than enzymatically active HRP, however, at 0.825 p.M
they were equally effective. The role of LPS in peroxi-
dase-induced MMC was investigated. As can be seen in
Figure 4, the presence of PB did not significantly affect
cytotoxicity by either HRP or DHRP suggesting that
observed MMC was LPS independent.
Since exposure of M#{248}to M-BSA was sufficient to
induce TNF, it was necessary to determine if it could also
induce MMC. As can be seen in Table 3, M-BSA was
more effective than DHRP for activating M#{248}to the
cytotoxic state. At 0.825 p.M DHRP and 0.485 p.M
M-BSA, both induced 20-40% cytotoxicity. Moreover,
there was no consistent effect of PB on MMC. Neither
Fig. 1 . Thioglycollate-induced peritoneal MO (10�) fromC57BI/6 mice were cultured in 8 x 50 mm tubes. After 30 mmincubation, the cultures were washed and exposed to one of thefollowing: control media, 0.04 pM of HRP, 0.2 pM DHRP, or acombination of HRP and DHRP. Opsonized zymosan and lumi-nol were added to the cultures. Light emission was measuredusing a luminometer which was programmed to record five2-mm counts. Each value represents the mean of duplicatecultures. The above data are representative of experimentswhich were repeated at least 3 tImes.
Fig. 2. Thioglycollate-induced peritoneal MO from C57B1/6mice were cultured in 8 x 50 mm tubes. After 30 mm incubation,the cultures were washed and exposed to one of the following:control media, HRP at 0.04 aM, mannose, or combinations ofHRP and mannose. Opsonized zymosan and luminol wereadded to the cultures. Chemiluminescence was measured usinga luminometer which was programmed to read five 2-mmcounts. Each value represents the mean of duplicate cultures.The data presented are representative of experiments whichwere repeated at least 3 times.
U fIR?
Q HRP+SPI1mM
0 HRP+SPI2mM
D HRP+SPI4mM150
100
50
0
80
60
40
. 8.2�tM-PB
B 8.2�LM+PB
U 0.82 p.M-PB
111 0.82p.M+PB
20
b.
HRP
1 50
D HRP
E
I-
z
zI-
. DHRP
0 DHRP+SPIImM
0 Dl-LRP+SPI2mM
0 DHRP+SPI4mM
1 00
50
HRP and M-BSA were also capable of inducing cyto-
toxicity in the MMC assay (Table 4). However, the levels
of cytotoxicity obtained (15-20%) were less than that
obtained with LPS sensitive mice either in the presence or
absence of PB . MMC was detected using concentrations
of HRP which did not induce secreted TNF (Table 1,
Fig. 4).
Previous reports from this laboratory have shown that
addition of polyclonal anti-TNF completely neutralized
HRP-induced TNF and significantly reduced HRP-in-
duced MMC [23] whereas normal rabbit serum used at
M#{248}-MediatedCytotoxicity 619
E
z
zI-.
a.200
Fig. 3.a,b: Thioglycollate-induced peritoneal MO from C57BI/6mice were cultured in 96 well microtiter plates. After 48 hrincubation, the cultures were washed and exposed to either 8.2p.M HRP or 8.2 pM DHRP, with and without sodium periodate(SPI). After 6 hr incubation, supernatants were removed andassayed on L929 cells for TNF. Values represent the mean oftriplicate cultures ± S.E.M.
c)
CC
c)
Fig. 4. Thioglycollate-induced peritoneal MO from C57BI/6mice were exposed to either enzymatically active HRP orenzymatically inactive HRP in the presence or absence of PB(100 ng/ml). After 6 hr incubation, the cultures were washed andmedia plus 3T12 target cells were added. Following 42 hrincubation, the cultures were stained using methylene blue andthe O.D. read at 660 nm. Percent cytotoxicity was calculatedusing 9 replicate cultures ± S.E.M.
TABLE 3. Effect of PB on MMC Induced by EnzymaticallylnactiveHRP and M-BSA
Treatment
+PB
P value
-PB
% Killing O.D. % Killing O.D. P value
ControlDHRPControlDHRPControl
M-BSAControlM-BSA
016C0
32d
0
17�0
20d
0.487 ± 0030b
0.408 ± 0.0240.484 ± 0.0240.330 ± 0.0200.487 ± 0.0300.402 ± 0.024
0.285 ± 0.0200.228 ± 0.008
<0.05
<0.001
<0.04
<0.02
0140
270
30
026
0.519 ± 0.0230.448 ± 0.033
0.5 1 0 ± 0.0 1 10.370 ± 0.0090.519 ± 0.0230.364 ± 0.013
0.287 ± 0.03 10.214 ± 0.004
<0.05
<0.001
<0.001
<0.01
aThioglycollateinduced M4 from C57B1/6 mice were exposed to media or 0.825 �aM of DHRP or 0.485
MM of M-BSA ± PB. After 1 or 6 hr incubation, the monolayers were washed and 3T 12 target cells with orwithout PB were added to the cultures. Forty-two hours later the cultures were stained using methylene blueand the O.D. determined at 660 nm using an ELISA plate reader. The % cytotoxicity was calculated using 9replicate cultures.bValues are mean O.D. of 9 wells ± S.E.M.COne hr induction period.
dSix hr induction period.
I-.
c)
CI-
C
I-.
c)
60
. M-BSA Anti-TNF 1:25
50 � M-BSA Anti-TNF 1:175
B M-BSA
40
30
20
10
620 Lefkowitz et al.
TABLE 4. The Induction of MMC by Exposing M4 From C3H/HeJ Mice to HRP andOther Glycosylated Proteins
Treatment Concentration % Cytotoxicity O.D. P value
Exp. 1HRP 0
2.7 j�M-
190.748 ± 0.015
0.598 ± 0.020 �0.00lM-BSA 0
1.5 �zM-
150.680 ± 0.252
0.580 ± 0.202 �0.0lExp. 2
M-BSA 01.5 �M
-
130.544 ± 0.0 160.472 ± 0.020 �0.Ol
G-BSA 01.5 MM
-
00.544 ± 0.0 160.553 ± 0.01 1 N.S.
aThioglycollateinduced M4 were exposed to either media, HRP, M-BSA, or G-BSA for 1 hr. After
incubation, the monolayers were washed and media plus 3T 12 target cells were added. After 48 hrincubation, the cultures were stained using methylene blue and the O.D. read at 660 nm. The % cytotoxicitywas calculated using 9 replicate cultures.
the same concentration as the polyclonal anti-TNF did
not statistically alter these parameters. Polyclonal anti-
TNF was added to cultures which were exposed to either
M-BSA or DHRP. In all instances, the presence of
neutralizing antibody to TNF markedly reduced MMC
whereas normal rabbit serum did not affect cytotoxicity.
The presence of a 1 :25 dilution of anti-TNF reduced
MMC induced by M-BSA from 51-4% (Fig. 5)
(P � 0.001). This concentration of antibody completely
eliminated secreted TNF. A dilution of 1 : 1 75 of the
antibody reduced MMC to 14% (P � 0.03). Similar
results were obtained utilizing DHRP.
DISCUSSION
Because M#{248}are exquisitely sensitive to trace amounts
of endotoxin, it was essential to identify its possible role
in these studies. HRP concentrations of 8.25 p.M con-
tamed approximately 1 ng of LPS/ml whereas DHRP at
this concentration contained 0.5 ng of LPS. Lower
concentrations of these reagents contained proportionally
less contaminating LPS. M-BSA at 0.825 p.M contained
0.3 ng LPS. The equivalent amount of LPS (� 1 .0 ng) by
itself did not induce TNF nor did it induce cytotoxicity in
the target-cell assay , as reported by others [13,14].
PB has been used by numerous investigators to elim-
mate the effects of LPS [30,33] . It has been reported that
PB interferes with the ability of LPS to induce superoxide
production [30,3 1] . Others have reported that reactive
oxygen intermediates (ROI), such as H,O2, enhance the
production of TNF [6,7]. It is known that superoxide
dismutates to H,O, which is a substrate for peroxidases.
Since PB inhibits superoxide production by LPS, the
reduction of superoxide could directly affect TNF pro-
duction. Other investigators have reported the presence
of two forms of TNF: membrane bound and secreted [9].
Only the former is required for cytotoxicity [9]. As
Fig. 5. Thioglycollate-induced peritoneal MO from C57Bl/6mice were exposed to 0.74 p.M of M-BSA alone, or M-BSA plusvarious concentrations of polyclonal anti-TNF. After 1 hr incu-bation, the monolayers were washed and fresh media plus 3T12target cells were added. Following 47 hr incubation, the cultureswere stained using methylene blue and the O.D. read at 660 nm.Each value represents the mean from 3 cultures ± S.E.M.
previously stated, diminished amounts of superoxide
could result in reduction of available H,O, which also
induces secreted TNF [5]. In the present studies, 100 ng
PB was effective in reducing TNF induced by enzymat-
ically active HRP but had no effect on either DHRP or
M-BSA-induced TNF. The fact that the addition of PB
succeeded in almost eliminating secreted TNF induced by
HRP could be explained by the fact that PB interferes
with radical production which may preferentially affect
the secreted form of TNF.
It is well documented that M-BSA and HRP bind to a
receptor on the macrophage surface [29] . The receptor
which binds these compounds is the MFR. Burton and
Gordon [4] reported that binding to the MFR induced
superoxide release from M#{248}.This fact, together with the
above data, suggests that 2 pathways leading to TNF
Peroxidase-Induced MO-Mediated Cytotoxicity 621
production may operate in this system� one sensitive to
PB (HRP) and another pathway insensitive to PB (DHRP
and M-BSA). The differences in sensitivity to PB could
reflect the levels of superoxide and H,O, production
induced by HRP and DHRP. The production of super-
oxide and/or H,O, can be measured using chemilumi-
nescence [36]. Chemiluminescence studies indicate that
HRP induced a respiratory burst (RB) [21] whereas M#{248}
exposed to DHRP and glycosylated-BSAs did not elicit a
RB. The inability to detect superoxide using DHRP and
M-BSA in our system could be the result of a relatively
short exposure time (10 mm). Other investigators deter-
mined superoxide production during a 1 hr assay [4].
Since HRP and DHRP are antigenically the same, it is not
surprising that DHRP was able to inhibit HRP-induced
chemiluminescence. It should be noted that mannose
inhibited chemiluminescence but only when it was
present in excessive amounts. M-BSA at the concentra-
tions employed did not inhibit HRP-induced chemilumi-
nescence. Rapid turnover of the MFR receptor could
account for the inability of low levels of M-BSA to
inhibit chemiluminescence (see below).
The MFR has been extensively studied 129,34,35]. It
is of interest that the MFR cycles rapidly every 10 mm
with only about 20% of the receptors being present on the
cell surface at any given time. This receptor is expressed
on resident M#{216}and decreases as M#{216}becomes more
activated I I .21. It is involved in triggering the RB and
phagocytosis. G-BSA also binds to the MFR but consid-
erably less efficiently than M-BSA [151. In the present
studies, the former induced 0-10 U of TNF and caused
minimal cytotoxicity (0-1 1%) in the target cell assay
using M#{248}from C57BL/6 mice. It is interesting to note
that neither BSA nor mannan alone induced either TNF
or MMC. This can be explained by the fact they do not
cause extensive cross linking of the MFR. It should be
noted that the rat MFR can be blocked by mannan
considerably more efficiently than the mouse MFR [25].
At this time specific antisera to the murine MFR is not
available to test this hypothesis.
Other investigators have reported that M-BSA failed to
activate rat alveolar M#{216}and mouse peritoneal M#{248}to be
cytostatic to L1210 tumor cells [24]. In these in vitro
experiments. the cells were pre-incubated for 24 hr with
M-BSA. In our hands, if M#{216}remained in culture with
M-BSA for approximately 6 hr. there was substantial
reduction in cell numbers due to detachment of M#{216}from
the microtiter plates. HRP induced peak TNF titers from
M#{216}6 hr after exposure to the enzyme. Present in vivo
studies indicate that intravenous (i.v.) injection of DHRP
induced peak titers of TNF in 45-90 mm with almost no
detectable TNF at 180 ruin (manuscript in preparation).
Therefore, it is possible that differences in the binding
affinities for the MFR of different preparations of gly-
cosylated proteins as well as kinetics of TNF production
could account for the conflicting results between the two
studies.
Sodium periodate enhanced the ability of HRP to
induce TNF but had no effect on DHRP-induced TNF.
Other investigators have reported that TNF production in
vitro was enhanced by the addition of H202 or a free
radical generating substance such as NaIO4 [6] . During
the 10 mm observation period, the presence of DHRP did
not induce detectable amounts of superoxide or H20,
using chemiluminescence. Binding of a ligand to the
MFR is known to induce a RB [4,15]. It is well
documented that H2O, is secreted during a RB [3] . At the
same time, ROI can inhibit ligand uptake by the mannose
receptor [5] explaining in part, the increased efficacy of
DHRP over that of HRP since the latter induced signif-
icantly higher levels of ROI. Therefore, it is possible that
there are both stimulatory and inhibitory effects of
radicals relative to TNF.
The highest concentration of sodium periodate (4 mM)
was inhibitory for both HRP and DHRP. This concen-
tration has been reported by others to inhibit TNF
production. The probable cause of the reduction was that
this level of sodium periodate caused damage to the cell
membrane [6]. The fact that radical generators affected
HRP but not DHRP induction of TNF implies again 2
different pathways of TNF induction: one resulting in
rapid radical production (HRP) and one with substantially
slower H2O, production (DHRP). These data, together
with the fact that the presence of LPS did not affect either
TNF induction or MMC induced by DHRP or glycosy-
lated-BSAs indicate that superoxide or H,O, may have
minimal effects in the PB insensitive pathway. There-
fore, a possible mechanism for M#{248}activation via an PB
insensitive pathway is the engagement of the MFR.
Moreover, although DHRP and HRP are antigenically the
same, it is possible that DHRP binds more efficiently to
the MFR than HRP. Enhanced binding to the MFR could
also explain the greater efficacy of DHRP as an inducer
of TNF and MMC. Studies to be published elsewhere by
these investigators demonstrate production of IFN� by
murine M#{248}exposed to HRP, DHRP, M-BSA, and
fucosylated BSA, but not other glycosylated-BSAs (sub-
mitted). These data taken in their entirety suggest that
cross linking of the MFR may play a role in activation of
macrophages by peroxidases.
Production of TNF and the requirement of TNF for
MMC by M#{248}exposed to HRP was proven using a
specific antiserum to TNF 123]. In the present studies,
neutralization of cytotoxicity with this antiserum mdi-
cates that TNF was also required for MMC induced by
either DHRP or M-BSA. This commonality underscores
the probability that TNF is an important mediator in these
reactions.
622 Lefkowitz et al.
Preparations of HRP, DHRP, and dialized HRP were
electrophoresed using polyacrylamide gel electrophoresis
(PAGE). Common bands were observed with all the
preparations; however, there was an increased number of
bands present in the dialized HRP. Extensive dialysis of
enzymatically active HRP used to stimulate M#{248},resulted
in an increased production of TNF, as well as increased
numbers of bands on PAGE. These results suggest that
“breakdown” products of this substance and/or exposure
of certain sites may be more effective than the intact
enzyme. A report in the literature indicates that multiple
bands associated with HRP increase after storage at 4#{176}C
[27].
C3H/HeJ mice are resistant to low levels of LPS. Data
obtained using these mice support the thesis that LPS was
not required for either peroxidase-induced TNF or MMC.
Both HRP and DHRP induced low levels of TNF using
M#{248}obtained from these animals. Levels of TNF were
usually 10-20% of those obtained from LPS sensitive
mice. It has been reported that a limited number of cells
from C3H/HeJ are capable of making TNF [19] resulting
in considerably lower levels of TNF. Cytotoxicity and
TNF observed using these M#{248}were always lower than
that obtained with M#{216}from either C57BL/6 or C3H/HeN
mice. The reduction in TNF and cytotoxicity could
reflect fewer cells which secrete TNF, a reduced produc-
tion of membrane bound TNF, or both. Since TNF is
required for MMC in this system [23] the lower level of
cytotoxicity obtained using C3H/HeJ mice was not
surprising. There was about a 3-fold difference in the
percent cytotoxicity obtained between the LPS sensitive
and resistant strains.
It is well documented that peroxidases function in
cytotoxic reactions [8, 18]. The present investigators have
postulated that these molecules function in the body via
activation of macrophages resulting in an increased
ability to eliminate aberrant cells and invading bacteria.
Further studies are required to show the immunoregula-
tory effects of endogenous peroxidases and identify the
mechanisms through which they operate.
ACKNOWLEDGMENTS
Anti-TNF was supplied as a generous gift from Dr.
George Gifford, University of Florida, Gainesville, FL.
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