Inhibition of natural killer cell activity in vitro by alcohols

Vol. 105, No. 4, 1982 April 29, 1982

BIOCHEMICAL

AND BIOPHYSICAL

INHIBITION ACTIVITY Sandra

S.

Department

Received

March

1,

OF NATURAL KILLER CELL IN VITRO BY ALCOHOLS -__

Ristow,

Department

RESEARCH COMMUNICATIONS pages 1315-1321

Jean

R.

Starkey,

G.

Michael

Hass

of

Bacteriology and Biochemistry University of Idaho Moscow, Idaho 83843 and of Veterinary Microbiology and Pathology Washington State University Washington 99164 Pullman,

1982

SUMMARY: The effect of the addition of small quantities of alcohols to cocultures of natural killer effector cells from c~IBL/~ mice and BDIX rats with The order of inhibition of NK cellYAC-1 tumor cell targets has been studied. mediated killing is 1-butanol ? l-propanol > 2-propanol > ethanol > methanol. The inhibition of killing due to the addition of alcohol correlates with deTherefore caution should be exercreases of effector-target cell binding. cised in interpreting results of cellular experiments in which these alcohols have been used to solubilize inhibitors.

INTRODUCTION Recently which

effector

their

tumor

much

inducers (1).

order

step an

that

phosphate,

in

effort some NC1 tosyl

i.e.

acetyl

tyrosine

dodecyl

lAbbreviations: sulfate.

to of

lyse

(5)

killer

cell

repeat the

to

and

serine

phenylalanine ethyl CM,

ester, complete

definition

natural

be

of

exceptional

is

affected cells

the

protease

the

T and

tumor

detail

serine

weight

may action

they to

to

cytotoxic

their

and

used

molecular

a proteolytic

served

In

have

large

In

3)

devoted

NK cells

since

(2,

laboratories of

(1)

been

of

cells.

cells

experience

has

populations target

surveillor feron

effort

mode

killer

of

by

cells

lyse

as and

previous

action

immune-

inter-

immunological

NK cells

several

and

protease

inhibitors

concerning

the

(4)

conclusions

(NK)l

interferon

without of

mechanisms

importance by

inhibitors

draw

of

presence

of

action. extend

some

protease

of

inhibitors,

chloromethyl must

these

be

medium;

1315

experiments i.e.

ketone dissolved NK,

natural

diisopropyl and

in

we

have

ob-

fluoro

substrate

analogues,

minimal

amounts

killer;

SDS,

of

al-

sodium

OOOS-29lX/82/081315-07$01.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol. 105, No. 4, 1982 cohols we

in

noted

protease

order

to

that

the

inhibitor

experiment

detailed

of

which

is

of

lower

alcohols

BIOCHEMICAL disperse

them

alcohol

alcohol.

below

have

in

control

plus

inhibition

AND BIOPHYSICAL media

for

RESEARCH COMMUNICATIONS

assay.

In

inhibited

NK killing

Therefore

we performed

which

showed

of

binding

of

on

NK cytolysis.

the

almost

exquisite

target

and

several

experiments as

much

a rigorous effects,

effector,

the that

small

as control

most

notable amounts

METHODS Mice and rats. C57BL/6 mice used in these studies were bred in the animal colonies of the Washington State University Veterinary School from pairs obtained from Jackson Laboratories, Bar Harbor, Maine. All animals used were males between six and eight weeks of age. BDIX rats were also bred at Washinqton State University from pairs obtained from Joseph Mayo, NIH, Maryland. NK Assay. Effector cell populations were obtained from C57BL/6 male mouse or BDIX male rat spleens by gently pressing them through a sterile fine screen into complete medium (CM) consisting of RPM1 1640, 10% heat inactivated fetal bovine serum, 25 mM HEPES buffer and 100 units/ml penicillin and 100 ug/ml Streptomycin. Erythrocytes in the cell suspension were lysed by exposure to sterile distilled water (mouse cells, 4 seconds; rat cells, 6 seconds). Spleen cells dispersed in CM were applied to a 10 ml sterile column of Fenwal scrubbed nylon fiber, type ZOO, to remove B cells and macrophaqes (6). After a 45 minute incubation on the column, the cells were eluted with CM, pelleted, and 5 x lo5 cells were dispensed into the wells of Falcon Microtest II plates. Rat spleen cells were not incubated overnight at 37OC to allow for "natural induction" of NK reactivity (7) since we wished to compare similar populations of effector cells for both species and such incubation is detrimental to mouse effecters. YAC-1 cells (obtained from Dr. J.M. Durdik, Fred Hutchinson Cancer Center, Seattle, WA) were labelled in 200 uCi of [51Cr1 sodium chromate in 1 ml of CM for 1 hour with gentle shaking, followed by two 30 minute incubations in large volumes of CM. They then were centrifuged, suspended at 2 x lo5 cells per ml, and dispensed into the test plates in 50 1-11 aliquots per well followed by the addition of the alcohol or CM control. As a control, effecters cultured in the presence of inhibitors were monitored for their ability to exclude Trypan Blue to ensure that the alcohols were not decreasing cell viability. Aliquots of the 5lCr labelled YAC-1 populations were cultured with the various dilutions of alcohols and their supernatants counted to measure toxicity of any alcohol to the target cells. After four hours incubation at 37OC in an atmosphere of 5% CO2 in air, the plates were centrifuged at 200 q for 10 minutes and Specific 100 microliter samples were removed and counted in a gamma counter. 51Cr release was calculated as follows: experimental maximum Percent

release

inhibition

release by of

2% SDS NK killing

Specific 51Cr release without inhibitor Specific 5 1Cr release

spontaneous spontaneous was

calculated

release release

x 100% from:

Specific 51Cr release the presence of inhibitor without the inhibitor

in x 100%

Alcohols. l-Butanol, l-propanol, 2-propanol, glycerol, and methanol were "Analyzed" Ethanol was the 95% reagent grade from Baker Chemical Company. ethanol 5% water distillate obtained from U.S. Industrial Chemicals, Anaheim, CA. The alcohols, in CM at a concentration of 4.8% (v/v), were added to the

1316

BIOCHEMICAL

vol. 105, No. 4, 1982

0:2 %

Figure

wells plates

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0'4

IO

SOLVENT

06

0 8

ADDED

TO

I 2

MEDIA

Butanol Inhibition of NK Activity of the C57BL/6 Mouse by Alcohols. (A-A); l-propanol (I-I); 2-propanol (9-o) ; Ethanol h-A,); and Glycerol (cl-a). Percent inhibition was calcuMethanol (o- 0); lated based on 16% specific chromium release from controls at the standard 5O:l effector-target ratio.

1.

of the microtest of l-2%, 0.68,

II plate to 0.3%, 0.15%,

achieve the and 0.075%.

final

concentrations

on

the

The target binding assay used was a modification of Target Binding Assay. the assay described by Roder (4). lo6 YAC-1 cells plus 4 x lo5 effector cells were centrifuged at 200 x q in 0.2 ml CM with 50 ~1 of CM (control) or 5% solvent in CM at room temperature for 5 minutes. The final concentration of alcohol in the incubation medium was 1% (v/v). The cells were placed on ice for one-half hour. Just before counting, one ml of Puck's saline G was pipeted onto the cell pellet and the mixtures were transferred to hemocytometers for counting. Results were expressed as the percentage of effector cells bound to YAC-1 targets. Statistical Methods. Raw data in counts per minute 5lCr from both rat mouse experiments were analyzed using an analysis of variance for a completely randomized design. This procedure was followed in order to determine whether the differences observed in NK inhibitory activities imposed by the presence the types and levels of alcohols were statistically significant.

As of

the

may

be

various

mediated

lysis

representative Analysis

of

all

differences

that

observed

in

alcohols

has

in

both

of

three

variance

the

RESULTS

AND

Figures

1 and

of

the

raw

between

significant

(P <

.OOOl).

1-butanol

> 1-propanol

data

order

Z 2-propanol

the

and

in

various of

the

counts alcohols inhibition

> ethanol

1317

presence on

performed

the The

2, effect

mouse

experiments

of

DISCUSSION

a profound C57BL/6

and

the

concentrations

The cells

minute and is

> methanol.

of

rat.

spleen

per

low

inhibition

BDIX

on

of

the

data from

of

51Cr

levels similar

In

shown

is

each

species.

reveals

over-

of

alcohols

in

both the

NK-

rat

are species: the

Vol. 105, No. 4, 1982

BIOCHEMICAL

%

Figure

2.

Inhibition

of

(A -A);

l-propan

Methanol culated standard

(o-o): based 5O:l

presence

of

methanol

addition

of

butanol

the

same In

by resent

both

to

three

nature Further

at

the

as

on in

NK

the

is

evident

of

the

level

that

.

the

Glycerol

effect

of

appears

to

the

produce

of the

alkyd

chain.

were

performed

of

target

do

of

decrease

to

is

Cells

order

of

cell

related

delineate

effector

the

NK

whether In

binding

to

C5 JBL/6

Mouse

Control Glycerol Methanol

10.7

+

1.3%

12.6 9.8 6.6 5.6 5.6 3.3

+ r 2 t + c

3.3 0.5 0.9 1.9 2.7 1.9

1318

YAC-1

repof

decrease

in lysis

in

the

length

and

the

effect

of

each

case

the

1

Bound

target

which

mediated to

cells.

of

results,

inhibit the

inhibition

or

recognition The

Furthermore,

1). order

affecting

alcohols

Alcohol

Butanol

but

performed.

TABLE

Z-Propanol I-Propanol

ac tivity,

were were

that

% Effector

Ethanol

MEDIA

Rat by Alcohols. Butanol (ol ); Ethanol (~-4,); Percent inhibition was calrelease from controls at the

mouse

alcohols

(Table the

experiments was

same

TO

the BDIX 2-propanol (n-0). chromium ratio.

effect

indicate cells

It

and Glycerol on 10% specific effector-target

experiments

parallels

species.

of

(a-r);

whether

binding

roughly

ADDED

RESEARCH COMMUNICATIONS

species.

discover

target

NK Activity

the

experiments, to

SOLVENT

little

both

cell,

lipophilic

alcohols

in

order

effecters binding

is

effect

effector

has

AND BIOPHYSICAL

Targets BDIX 17.8 13.3 17.5 12.7 8.1 9.4 6.4

Rat k t f fr k 2 +

3.2% 3.9 2.9 1.1 1.5 1.6 2.3

the level

Vol. 105, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL TABLE

With

Treatment Alcohol,

of Target Followed

1)

Control (sham with complete

2)

Pretreatment 1.2% methanol

butanol,

the

least

amount

of

ethanol

and of

in

alcohol

the

after

of

the

wash

methanol,

different The

by

of

the

cells

and

we

At

higher

be

extracted

shown

that

treated

from 2.5%

the

are

alcohols

and

coworkers

of

+

17.2%

+

+

14.9%

-

+

18.2%

+

-

14.9%

+

+

16.6%

+ -

-

16%

+

16.3%

+

+

16.8%

+ -

-

13.1%

+

16.4%

+

+

16.4%

+

-

15.8%

-

+

15.7%

+

+

15.7%

is

than

membranes. of

butanol

selected

were

in data

that

shown

in

profoundly

those For

in

example, are

capable

1319

in

increase

effect used

is

killing.

alcohols

principal

disappear.

presence

these Le of

not

is

and

extracting

this hour

performed that amount

statistically

an

NK assay

of

It

has

suq-

operating

experiments, Grue

For

The

membrane

which

case

one

assay

being

the

2 indicate

combinations

alter

In

for

the

Table

effects

the

1 as

assay.

incubated

2 and

washed

figure

tested.

Table

inhibitory

that

the

concentration

effecters

and

from

in

highest

show

the

was

inhibition

The

(8,9)

alcohol

solutions

the

the

may

this

cell

15%

-

and

presented

of

that

-

100%

CM.

in

which

levels

in away,

control.

speculate

+

shown

washed

the

Lenaz

14.8%

used

targets

concentration

quantities

qested

was

the

seen

data

1.2%

at

is

from

give

8 Specific Chromium Release

On: Targets +

2-propanol to

the

alcohol

NK activity

small

necessary

experiments,

one

when

and

Treatment

+

with

l-propanol,

series

with

Pretreatment .3% butanol

6)

of

with

Pretreatment 0.6% 2-propanol

5)

treated media)

with

Pretreatment .6% 1-propanol

4)

and C57BL/6 Effector Cells Effect on NK Activity by Wash.

with

Pretreatment 1.2% ethanol

3)

2

Solvent Effecters

Treatment

RESEARCH COMMUNICATIONS

been fluidity,

here. proteins

coworkers tumor

may (10)

antigens

have

Vol. 105, No. 4, 1982 from

cells

levels

without

in

our

throughout it

assay,

surface

short

time The

in

our

proteins

as

of

the

93%

Chanq

the

which

used

yet

alcohols

should

be to

when

NK levels

returned

the

would

used

alcohols

alcohols

to

since

However, were

to

alcohols

have

approached

of

2-propanol

dry it of

in

NK

present

normal

after

extracts

any

been

resynthesized

have

observed

that

washing,

critical

the our

in

at

a

two

results

where

inhibitors,

or

this

noted

for

level.

that

inhibitors

may

have

pure

(v/v).

on

NK activi-

dramatic

effect

and

indicate

that

substances

be

seen

0.5%

the

soluble

over

We have

below

these lipid

shown). mechanism

accounts

alcohols

species

at

diisopropyl

negligible

illustrate

not

We have

for

only

the

solubilize

which

data

probing

1 mM level

are

not (data

caution.

choice

effect

well

drugs

to

the

of

in

the

substrates,

at

operates

as

NK activity

NK cytotoxicity

interpreting

12:l

with

effects

defined,

assay

for

a solvent

of

the

needed

media

inhibitory

completely

solubilize

and

be

mechanism

on

25:l

should

since

have

in

solubilizers

(11)

complete

at

left

as

disperse

Eisen

in

but

alcohols

inhibition

and

Although not

capacity.

depressed

with

these

of

amount and

sulfoxide

is

the

ratio,

the

dimethyl

ty

since

effect

cytotoxicity hands

did

only

because

inclusion

of

were

RESEARCH COMMUNICATIONS

span.

fluorophosphate half

tumoriqenic

treatment

effector-target

The natural

brief

depressant

5O:l

of

and

that

AND BIOPHYSICAL

their

experiments

unlikely

cell

the

destroying

the

is

BIOCHEMICAL

have

caution been

substances.

ACKNOWLEDGMENTS This work was supported by NIH grants RR 07170 to the University of Idaho, the Animal Models Program of Washington State GM 22748 to G.M.H. and RR 00515, University. Ristow in the Department Correspondence should be directed to Dr. 115 Agricultural Sciences Building, Univerof Bacteriology and Biochemistry, This work is published with the approval sity of Idaho, Moscow, Idaho 83843. of the Director of the Idaho Agricultural Experiment Station as Research Paper We acknowledge the capable assistance of S. H. Tarnq who performed No. 81510. the statistical analysis for this paper and the expert secretarial assistance of Carol Borden who typed the manuscript. REFERENCES R.

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