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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