Effectof interferon-y

Masumion human

Aoki,

The Second Department

neutrophil

Yasuo

Ono and Otohiko

Itabashi-ku,

Tokyo 173, Japan(Received March 30, 1992-Accepted

studiedmetabolite
1,000

thegenerationor

10,000dependentofneutrophils

with
for

increasehumanofTherewasresulted

nosignificantincubation

withtheTheincubation

at

37•Ž

significantly

by

Nc-5.

Preincubating

IFN-y

alone

enhancement

during-induced

CLfor

4 hours.

fromelderlypatients

wasto

that

obtainedfromhealthy

young

IFN-y

activatesthat

therapy
IFN-y,

Interferon- y (IFN-y)forneutrophil

functionintractable

infections

Chemiluminescence,

activates

monocytes

humanandin

Hospital

results have been reporteds-7)

generation of oxidative metabolites in inflammatory
cells. In this study, using CL, we examined theeffect of recombinanthuman IFN-y(IFN-y)invitro on theobtained from

respiratory
burstof neutrophils
healthy young adults and elderly

patients, and speculated on the use of IFN-y
immunotherapeutic
agent.
Materials
Subjects
Eighteen healthy
medianas anrange(10 mens, 8 womens,25-36)

andnineanda 1.75-foldneutrophilwithwas.

IFN-y.

also

enhancedtheeffectofAnincreaseobserved.
beofIFNin

CL

These findings

clinicallyusefulin

im-patients.

patients(4 mens,admittedto Teikyo

5 womens,

medianUniversityage

78.8

years,
range 70-89)
were studied.
All elderly
patients had been admitted with neurologic or heartdiseases without acute clinical conditions,
glucoseintolerance or elevation of serum creatinine levels.
Chemicals
Dulbecco'sTokyo)

and media
modifiedcontaining

0.3 g/1 at pH 7.4

Eagle'smedium(Nissui,

25 mM HEPES and L-glutamin(MEM)

was used to dilute sam-ples. Luminol (Tokyo Kasei Kogyo, Tokyo) andzymosan (Sigma Chemical Co. ) were dissolved inphosphate
25 mg/ml,

human

age 29.2 years,CLbuffered saline (PBS) at 20 jig/ml andrespectively.
Phorbol myristate
acetate(Sigma Chemical Co.)sulfoxide(DMSO) atand Methods

volunteers

mayofunits/ml

neutrophils

elderly

Measurementof chemiluminescence(CL) isthought to be a reliable tool with which to study thewaselderly

andreported that neutrophils primed by IFN-y haveenhanced functional activity2-4) However, in somephorbol

incubation

stimulateadditionwithzymosan,

preincubation

In

blood

1,000

neutrophils

oxygenluminol-hours'

notevaluatedadultsmacrophages'.
Less focus has been given to themodulation of neutrophils by IFN-y.
It has beencases, contradictory

of

whole

withdida 2 hours'

obtainedmunosupportive

IFN-y

Four

theenhanced

in zymosan-induced

However,ofofneutrophils

increaseon

non-opsonized(lumispheres)
units/ml(IFN-y)vitro.

aeruginosa

CL.

yin

neutrophils

comparable

1,000

interferon-stimulated

CL.

August 5, 1992)

4 hours

in a 1.16-foldywords:

forPseudomonas

aeruginosa-induced

after

Keyyenhanced

microspheresuggestneutrophils

IFN-orIFN-y

4 hoursin P.

human(CL)(PMA)

Luminol-bindingon

recombinantchemiluminescenceacetateCL.

ofunits/ml

myristateIFN-yeffectKunii

of Internal Medicine Teikyo University School of Medicine,

2-11-1 Kaga,We

chemiluminescenceinterferon-was dissolved in dimethyl
100 pgiml.
Recombinanty (IFN-y)

was kindly provided

by Shionogi Pharmaceuticals,
Osaka , Japan, andlumisphers were purchased from TORAY Industries

CHEMOTHERAPY

1384

Inc.,

Tokyo,Japan.

Isolation

andpurification

Neutrophilsvenouswerebloodresolvingon(10 IU/ml

mediumland).

Blood

layer

atroomin

Growthofwas, andonce

300 x gforThe

neutrophil

with

MEM,

andbacteria
aeruginosaNc-5

on nutrientheart

infusionwerethen

twice

and

washeda concentrationofIncubation

aboutwithinoverlaid

attemperature.
washedScot-

carefully

centrifugednightstoredmono-poly
Labs.,

MEM.

Pseudomonas

Purified

heparinizedusing
Flowml)withdrawn,

resuspendedfrom(M-PRM,ml)

was

neutrophils

blood)(3.5-7

M-PRM

30 minutesofprepared

was
broth

grown

over-

at 37°C.

Cells

resuspended

in MEM

towas

portions

atdiluted

insterilewater

concentrationsof

and1,000,000units/ml

at -80•Ž;aliquots

were

not

re-frozen

after

thawing.

Anwas

dilutedwithnormalsalinetoa finalconcentrationor100,000units/ml.

Whole(5 x 105/ml)

were

blood

incubated37•Ž
allaliquotwithforincubatedof IFN-yconcentrations

lengths

ofwholeblood

forserved

asml

CL0.1

added

to

round-bottomed

ofaddition

of

0.96

luminolandincubatedabsenceofIFN-yzymosan(500ƒÊg,aeruginosa

added
Theml.

CL

suspen-in theabscencemeasuredusingGermany)

assay:

(i)

ofaat

In

containingatin37•Žpl).

Non-opsonized

PMA(0.5ƒÊg,(ii)assayedin theP.

aeruginosa-induced

cells

wereadded

pooledserum.

the10' bacterial
human

Lumisphere-induced100ƒÊl)was

samewayor

5ƒÊl)
was

or
then

Neutrophil
5 x 10cells/

asdescribed

inCL

assay,

2 x

to samples

CLofpresence,measured.

the20 pitheconcentrationwasby(10(108 cells,

wasWholeblood

followed

MEM

20CL

wholetubes

mlneutrophil
wasofInCo.,

heparinized

Nc-5

and

CL:10 ill

atassay.

30 minutes.

CL:(2)was(Berthold

Luminol-dependent(i).

IFN-ytoneutrophilperiods

(CL)
9505

bloodP.

orofmeasurements

LBfor

(1)

priorcontrols.

Chemiluminescence

37•Ž

time

identical

Chemiluminescence

Biolumat

10,000neutrophilsvariousvariousexperiments,

sions

ofor

assay:

containing

Samples

1992

containing 5 X 105 neutrophils were incubated, then20of lumispheres were added and CL wasmeasured.
Data presentation
The integral CL over the whole period (30 minutes) was calculatd and IFN-y-incubatedcells
were compared to IFN-y-nonincubated(control)cells. Results are expressed as increased rate of theintegral CL, that is the ratio of CL of IFN-y-incubated cells to that of the control.
Statistical analysis
The statistical significance of differences between
results was calculated by Student's t-test.
Results
Stimulus-induced CL in whole blood obtained
from healthy young adults was increased comparedwith control values when whole blood was tested
after 4 hours' incubation in the presence of morethan 1,000 units/ml of IFN-y. During a 2 hours'
incubation under the same conditions, there was no
significant enhancement. As shown in Fig. 1, preincubating whole blood with 10,000 units/ml of IFN-

109 cells/ml.

IFN-yIFN-yDEC.

y for 4 hours resulted in 1.10-1.13- and 1.53-foldincreases in zymosan-, PMA- and P. aeruginosainduced CL, respectively. The CL response to 1,000
and 10,000 units/ml of IFN-y treated neutrophils
was similar. A dose-dependent response of luminol-enhanced CL was observed when neutrophils wereincubated with IFN-rat various concentrations (10-1 ,000 units/ml) for 4 hours prior to the addition ofP. aeruginosa (data not shown) . Four hours' incubation of neutrophils with 1,000 units/ml of IFN-yenhanced CL 1.75 fold (Fig. 2).
Preincubation of neutrophils with IFN-y for 4
hours resulted in a 1.16-fold increase in zymosaninduced CL (Fig. 2). Significant enhancement was
not observed with PMA, which differed from theresults of whole blood CL, but the peak time wasslightly reduced (Figs. 2, 3).
Lumisphere-stimulated
CL was also enhanced
after incubating neutrophils with 1,000 units/ml ofIFN-y for 4 hours (Fig. 4).
The addition of only IFN- y did not stimulate
neutrophil CL (data not shown) . Also, during a 2hour preincubation with IFN-y.
Furthermore, comparing the effects of IFN-y on

VOL.40

NO.12

IFN-y

and human

neutrophil

BAFig.

1.

EffectWholeofblood

IFN-yonluminol-dependent

incubatedwith(•œ-•œ)(B)

Pseudomonas

andare mean*p<0.05,ofvaluesIFN-y.

±2.

Effect10,000byzymosan

(A),

PMA

(C)

measured.

errors

wasof

6-16

Data

separateofIFN-yon

luminoi-dependent
wereexperiments.

neutrophil

incubatedofPseudomonas

aeruginosa(C)

presented

aremean±standard

errorsexperiments.
*p<0.05,**p<0.01

hours

increased

neutrophil CL obtained from healthy young adults
with those from elderly patients(CL was assayedin the same way),

we found that the increasedof CL in the latter

was slightly

but the difference(Fig. 5).

ratehigher than that in
was not statistically

Discussion
The results presented in this report demonstratethat preincubating
neutrophilswith IFN-y for 4

CLunits/nil

inducedoxygen.
haveCL.

100andvaluesIFN-y.

with(•œ-•œ)(B)

presented

C

units/ml

PMA

the former,
significantorinduced**p<0.01

suspensions

1,000

CL.

(A-6)

B

Neutrophil
orblood

units/mlCLstandard

whole

1,000

aeruginosa

AFig.

C

was

units/ml

1385

chemiluminescence

by
was
of

Data

5-18

theirshown(A),measured.

Previous

separateability

tostudies

that

IFN-y

functionssuchasactivities

andfungalreportson

IFN-y

study,

using

CL,

vates

neutrophil

metabolites(A-A)

zymosangenerateotherenhances

oxygenhave

someneutrophil

metabolism,

phagocytic
However,

beensome

conflicting5•`7).

demonstrated

thereactive
investigators

killing2-4,8-12).

function
duringbythatThis

IFN-ytogenerate

phagocytosis

ofactioxygen

non-opson-DEC.

CHEMOTHERAPY

1386

1992

AB

Fig. 4. Effect of IFN-y on lumisphere-induced
neutrophil CL.
Neutrophil suspensions were incubated for 4
hours in the presence (-)
or abscence(-)
of 1,000 units/ml of IFN-y. Data
presented are mean values obtained from 7
separate experiments.
cpm; counts per minuteCBA

Fig. 3. Time course of IFN-y enhancementofluminol-dependent
CL.
Neutrophil suspensions were incubated for 4
hours in the presence (-)
or abscence(-)
of 1,000 units/ml of IFN-y. After theadditionof zymosan (A), PMA (B) andPseudomonas aeruginosa (C), CL traces wererecorded. Data presented are mean values
obtained from 12 separate experiments.
cpm; counts per minute

Fig.

5.

Comparisonoffrom

Our results

healthy

young

NeutrophilwithinducedbyWe also demonstrated

that IFN-y

increased

int-The additionof IFN-yphil CL per se, and IFN-y

did not stimulaterequiredobtainedandelderlyNS;

timeunits/mlzymosanmean(A)wastheandCL

Pseudomonaspresent-standard

errorsfrominhealthyyoung

adults6-8experiments

in6

elderly(•›-•›).
notsignificant

to

exert

an

effectthan

latingfactor

(CSF)

ortumor(TNF). Theseresults

al.14)and

in-IFN-y.

Data±

fromwereofmeasured.

valuesexperimentsand(5•~105/ml)

1,000(B)patients

tion

racellular
reactive oxygen levels during phagocytosis of luminol-binding
microspheres (lumispheres).

are(•œ-•œ)

increased luminol-dependent
CL induced by preopsonized Staphylococcus aureus and enhanced the inikilling.

onCL

adultssuspensions

cubated-18

aresimilar to those of Edwards et al"), who reported
that the preincubationof neutrophils with IFN-ytial rate of bacterial

IFN-ypatients.

edand P. aeruginosa.

ofneutrophil

aeruginosa

ized zymosan

effectsluminol-dependent

Perussia

neutro-tionofa longer incuba-wasnecessaryetneutrophils
foragreeal.15)

witheithercolonynecrosisthose, whoshowedwithIFN-yfor

thepotentiation

stimufactorofBerton

etthatincuba-severalhoursofsuperoxideVOL.40

IFN-yNO.12

generationor CL response.
action of IFN-y
may differ
cytokines.

It is possible

and human neutrophil

The mechanism
offrom that of otherthat4)

studies5)7)

IFN-yinchronicrecognized").

This finding

was basedfrom patients with CGD21.22). In the elderly, reduction in neutrophil function, such as superoxide pro-D

A,

that IFN-y

activates

neutrophilfunction[letter].

Ann

Steinbeck

Mby P. aeruginosa)
production

in elderly

patients

Melby

1)

Nathankine

that

activates

metabolism

and158:

Shalabyhuman

670•`689,
MR,

Activationofhumanfactors.
Ton
icity
areY,

enhances
whenused.

Rubinlympho-B14)

J

B
B

B,
S,

H:

GrowthE,M

polymorphonuclear

J ImmunolW

suboptimal
ImmunolJ

A:A,

Cappelli

M,

Dusi

S.

250•`251,

1988

Neutrophil

activationRev

Infect

Dis

R,

Della

BiancaBertonhuman

andexocytosis.

Midtvedt

T,G:

11: 549

V,

Interferon-neutrophil

oxygenImmunology

63:

Degre
on

M:

Effectphagocytic

499

of

human

activities

leukocytes.

BlanchardDinhibitionof

APMIS

(B)

FriedmanK,

HalkiasD,ofCandidaalbicans

polymorphonuclear

90:

by

interferon-yand

137:

2980•`2984,Morrison

C J,

Brummer

activationof peripheral
byInfectKowanko

I C,

by
62:

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In vitro

resultsA:infungal1989

Stimulation

andoflysosomal

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interferon-gamma.

Immuno-

1987
Say

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thekilling

HughesGammainter-of

Staphylococcus

aur-J

Gen

134:

neutrophils.

Zeni

L,

is

MicrobiolM

ablemetabolism

ofBiophysV:

Cassatellainterferon

BiochemD

57: 2953•`2958,

burst

human

BertonStevens

interferon

Ferrante

S W,

1988

E,

Immun

human

37•`42,

factor.

gamma

enhancesbynecrosis

1986

polymorphonuclear

149-151,

Edwardstumor

activa-blood

respiratory

byneutrophils:

ImmunoloxidativeExp

Rinderknecht
Plladino

interferon-gamma

Smith

Roth

leukocytes

109:

•`Y,

Gamma

oxidative

activity.

K,

and

135:

2069-2073,

Merigan

T C:

antibody-dependent

Cell

E,
the

R J: Interfer-Res

A,

toRossiF:enhance

human

the

neutrophils.

Commun138:

Kobayashi

M,

M

G: Immuneinterferon

1276•`1282,

1986

FinklebyBashamM
asmacrophage

AggarwalP,

necrosis

Wiebe

Hamill

1982

J

logy

1983

L

functionsW,

interferon-y

antimicrobial

Svedersky

phil3)

HofJ,

Med

interferon

DjeueusMurray

Identification

Med2)

C F,Y:

44•`•`K,release

Citedan179:

1988

feronLiterature

InternMkilling.

is decreased.

M,cytokines.

neutrophils13)

ofgranur-cytokines:

Dactivates

philwhen IFN-yActivation

1989

Cassatellationoxygen production)and may be clinically useful inthe control of intractable
infection (e.g., mediatedN:

polymorphonuclear

recombinanthuman12)(reactiveMusherand181•`184,

duction, phagocytic activity and bactericidal capacity, has been reported22,23). Our findings suggest

U

Immunobiology

polymorphonuclear11)

Im-1989Watson506,

showing that rIFN-y increased the superoxide production and bactericidal
activityof phagocytesofJ

neutrophilic

immunomodulating

metabolism

on studies

Riedestudy.

gamma10)anti-1981byleukocyte

granulomatous disease (CGD), in which the abilityof phagocytic cells to generate reactive oxygen islackingor markedly
impaired,
hasbeenthe(ADCC)

leukocytes.

A,ocytes

Grzoskowiak9)

enhances

cytotoxicity

Kapppolymorphonuclear

568,

neutrophil CL response in the elderly was similar tothat in healthy young adults. Recently, the clinicalrecombinant

G,

human

by

production is decreased in the elderly18.19), but thisstudy showed that IFN-y-induced
enhancementofInterferon

1585•`1588,

on-gamma8)

of

127:

Zeck-kapp

55,

Little is known about the impact of aging on thefunction of IFN-y.
It has been reported that IFN

efficacyK:cellularultrastructural

are neces-sary to elucidate
the intracellularmechanisms
underlying this increased neutrophil function.

Bergpolymorphonuclear

munol

synthesis,
receptors

6)

AdditionalP,

human(FcR high) on the neutrophil surface or alterationof the NADPH-oxidase
system14-17). However, themolecular basis of the mechanism
has yet to besufficiently clarified.

Hoklandbody-dependent

the effect of IFN-ydepends on RNA and de novo proteinincreased
expressionof high-affinity

1387

chemiluminescence

cellularconcentrations
88: 393•`400,A15)Jr.:

Perussia

B,

Trinchieri

neutro-tional

tumor

Fc

1985

properties

ofreceptors

andnecrosis

factor,Interfer-colonystimulating

cytotox-774 ,of antibody
198416)Rossi

humanE,

granulocytes:

rolltumorofandgranulocyte-macrophage
JI,
func-lymphotoxin,

effectfactor.

Anegonenhances

Immunol

138:

of

765•`

1987

HumphreysJ

M,

StimulasionofproteinHughessynthesis

V,in

EdwardsS

humanneutro-W:

CHEMOTHERAPY

1388

phils

byyinterferon,1241•`1246,
17)

Newburger

PJ,binterferon-y.
1988

Rytel

M

21)

S

J

viralofinterferon.

JohnI

G:

in

InfectDisphagocyte

153:

RinshoSeijinbyo
in

1991
RS.

USA

85:

Turnerand5215•`

A,
23)

feron

gamma.

Sechler

J

1988
of

InfectDisN

M

patientsG,

Englphagocyte
granulomatous
Natl

Nagel

J

Acad
E,

A,

Dinauer

M

P

PartialE:

Recombinant

C,

Jaffe

HS.

correction

1988

J,

J

patients

polymorphonuclearofFJ,

1988
Adler

bactericidal

leuocytes
adults.

withchildhood.

4784•`4787,

ChrestandJ

Emanuelli
AnfossiOrkincyticoftologythefromnormal37:529•`534,

GerontolG,
G,

LanzioM,

Calcamuggi

activity

inT,

Influenceleukocytes

inhealthy

32: 308•`316,

AnfossiG:

human

Romanoof
vitro

subjects.

ト好 中 球 の活 性 酸 素 産 生 に お よ ぼ すrecombinant
検 討 した 。 全 血 を1,000まaeruginosaNc‑5刺意 に増 強 し た 。 分 離 好 中 球 とIFN‑γ
好 中 球 をIFN‑γ1,000units/mlと4時
激 で1.75倍

激 後 のluminol依を4時

humaninterferon‑γ(IFN‑
myristateとともにacetate(PMA)ま存 性chemiluminescence(CL)が間 保 温 し た場 合 もCL反間 保 温 後,zymosan刺の 有 意 な 増 強 効 果 が み ら れ た 。 しか し,IFN‑γ

た 。 ま た,2時

乙彦

た は10,000units/mlのIFN‑γ

37℃ で4時 間 保 温 す る と,非 オ プ ゾニ ン化zymosan,phorbol
た はPmdomoms

有応 を増 強 し た 。 す な わ ち,

激 で1.16倍,P.aeruginosa刺自体 は 好 中 球CLを誘 導 しな か っ

間 以 内 の 保 温 時 間 で は 有 意 な 増 強 効 果 は み ら れ な か っ た 。Luminol‑bindiugmicrosphere(lumisfere)を用 い た好 中 球 内CL反時 間 保 温 後 に 有 意 な 増 強 が み られ た。 さ ら に,高応 に お い て も,IFN‑γ1,000units/m1と4
齢 患 者 の 好 中 球 に お よ ぼ すIFN‑γの影 響 に

つ い て も検 討 し た と こ ろ,健 康 成 人 と同 様 のCLの増 強 が み ら れ た。 こ れ らの 成 績 か ら,IFN‑γ が 好 中球 機 能 を活 性 化 す る こ とが 示 さ れ,臨 床 的 に,高 齢 患 者 の 難 治 性 感 染 症 に お け る免
疫 補 助 療 法 と して 有 用 とな る可 能 性 が 示 唆 され た 。
*東 京都 板 橋 区加 賀2‑11‑1

H:

S,

age
phagoGeron-1986の ヒ ト好 中 球Chemiluminescence

青 木 ま す み ・斧康 雄 ・国 井
帝京大学医学部第二内科'

我 々 は,ヒW
capacityにお よぼす 影響γ)の 影 響 をinvitroでI:

reconstitutes
in85:

Gallin198224)

13:humanINF‑γ146•`451,

USAS,

polymorphonuclear

Newburger

319:
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aged

X-linked

MedH

Sci

1992

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young

management

indefectiveofmodulation

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Oxidative

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Recombinant

bymitogens

younganddisease.

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to984•`987,

Interferon-yC,phagocyte

expressionScielderly

S: Production

973•`978,H,38:

Whitney
ofresponseMorigranulomatousSK

antigens

A,

gene

Acad

Larratt

chronic

Ezekowitz

Natl

Interferon

I,

R
Induction

chain

K:

J

Sugawara

H:heavyW,subjects.

20)

Ezekowitz

Proc

5219,

Kalbfleisch

19)

E,

Orkin

cytochrome

andPharmacol

1989

Wright18)

BiochemDEC.

on