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 Macrophages

(M#{248})play

a central

role

in the

combination “cytotoxic

initiation

of both cellular and humoral immunity I 1 ] . A number substances have been shown to activate M#{216}to

of the

cytotoxic

li-

state

in

popolysaccharide

and

vitro

(LPS)

a number

including from

interferon

gram

of carbohydrates

negative

[28]

organisms,

including

bovine serum albumin M#{248} are a major source

and fucoidan [16,17]. of immunoregulatory

such

(IL-l)

as

interleukin-l

(TNF)

[9].

TNF,

a

In addition

number

of

and

to the

y,

tumor

maleyl Stimulated substances

necrosis

immunoregulatory

investigators

have

factor

described

of the

importance of TNF in macrophage-mediated (MMC) [9, 12] . Because of the various

secretory

ities and probably

M#{248} these cells defenses against

cancer

cytotoxic represent

by

of activated the major

cytotoxicity

capac-

[1,2].

Previous shown that tors. zymes

functions one of

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,

mammalian has

been

in

cells shown

the regression

and a halide ion, of killing bacteria,

[8,18].

Horseradish

to generate

free

of Novikoff

We have reported posed to HRP, myeloperoxidase vitro

role

with H207 triad” capable

form a potent viruses, and

peroxidase (HRP) [ 10] and cause tumors in rats [ 1 1].

radicals

hepatoma

that thioglycollate-induced M#{248}exlactoperoxidase, microperoxidase, or secreted TNF and became cytocidal in

as determined

by a target

cell

assay

The present studies were undertaken enzymatic activity of peroxidases was

[20,23].

to determine if 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 enzymatic induced Received

reactions.

January

Reprint

requests:

Sciences,

P.O.

79409.

The

present

activity nor MMC. However, 7. 1991 Doris Box

4149,

;

L.

studies

show

that

LPS was required TNF production accepted Lefkowitz, Texas

Tech

neither

by

HRPM#{248}ex-

Department

of

Biological

University.

Lubbock,

February

for

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

CeH/HeN,

were obtained Laboratories

from (Bar

ME).

Reagents HRP

type

V 1,

inactivated

horseradish

Co. (St. Louis, MO). Activator solutions immediately prior to use and filter sterilized

were prepared using a 0.22

jim

MA).

filter

(Millipore,

Bedford,

Dulbec-

DMEM. Phosphate-buffered as described previously

DMEM preparations ma) at a concentration anti-TNF

was

antibody does a 1 :25 dilution ml as assayed

Assay

were of

saline

[ 1 8]

supplemented 100 ng/ml.

from

react with lymphotoxin [14] and greater than 250 U of TNF/0. 1 cells.

All solutions were Limulus amoebocyte

George

Gifford.

This

Activity tested for endotoxin activity lysate assay (Associates

of

using a Cape

Cod, Woods Hole, MA). Solutions containing 8.2 HRP contained 1 .0 ng LPS. All other media reagents used contained 0.5 ng of LPS/ml.

jiM and

Thioglycollate-induced as described

previously

peritoneal [22].

Briefly,

M#{248} were

collected

mice

killed

were

by

hundred each

microliters well,

and

at 37#{176}C.Non-adherent cells twice with 100 p.1 of DMEM. of complete

the M#{216} were

DMEM

incubated

were 48 hr prior

added

by staining

to

to use.

to the

with

200 pA of PBS. Two in 100 mM NaH,PO4 Absorbance of the solutions

cells.

a microtiter as follows.

first

well

of

50 pA of 0.033%

determined neous where

using

the

(MMC)

3Tl2

cells

were

assay.

One

used

as

and

blue

with

target

cells 6 hr and

at an

in 10 mM

% cytotoxicity

.

.

and

a microtiter M#{248} was

DMEM incubated

borate

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

the TNF

of complete DMEM cells were added to the

washed with borate and allowed to air

containing was

effec-

incubation, saved for

the cells were fixed in 10% for 10 mm, and stained with

0. 1 N HCI,

at 660 nm using wells containing

previcultured 100 p.1 of

were added to each without peroxidase.

of 16: 1 . After were aspirated

At this time formalin

methylene

wells

simulta-

as described collected incubation.

wells. Another 100 p.1 of complete to each well and the cultures were

The plates were unbound stain,

toxicity

assayed

hundred microliters 6 x l0 3T12 target

appropriate were added

two

ax + b were solved and below the theoret-

the peroxidase received DMEM

cell ratio supernatants

extracted

was

Briefly, M#{216} were above. After 48 hr

tor:target culture containing

=

Assay

DMEM containing well. Control wells NIH

formula:

form y above

were was

and x = the reciprocal of the correspondThen 0.50 was substituted for y and the calculated and expressed as U/l00 p.1.

ously [23,32]. as described

the

following

equations of the y = % cytotoxicity

ical 50% point ing dilutions. TNF titer was

hundred microliters of were used to lyse the was measured at 550

plate reader. The TNF titers The percent cytotoxicity

3T12

cells

and

the

cyto-

as follows:

r

were Two

added

with ethanol

0.5%

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 removed by washing

twice 50%

another 42 hr. phosphate-buffered

Collection

MO

hr at 37#{176}C, followed

Cytotoxicity

obtained

were

neutral red (w/v) in PBS. After this incubation, cells were incubated for an additional hour at 37#{176}C,and washed

MO Cytotoxicity

with PB (SigRabbit polyclonal

not cross neutralizes on L929

for Endotoxin

Dr.

(PBS) Certain

.

to be tested

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

nm using calculated

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

of the samples

20

peroxidase

(DHRP) (