CHEMOTHERAPY

540

Effect of recombinantchemiluminescenceIsaogranulocyte

Secondofof human

Ohyatsu,

Yasuo

Department

The
the

effectfunction

cence
of

of

recombinant

of

human

(CL)

TeikyoUniversity

Tokyo

173,

Japan

December4,

1992•EAccepted

January

14,

1993)

in

terms

human

investigatedPMNs

stimulation

with

PMNs,

comparedthepowerfulpriming

granulocyte-macrophagerhG-CSF

on

the

same

as

of

rhG-CSF

at

lower

in

Incubationof

rhG-CSFfromhealthy

adults,at

non-opsonizedwithonzymosan,theresponsetheCLcolony-stimulating
PMNs

obtained

from

in

PMNs

on

activated

PMNs

and

at

levelsobtained

weak

from

healthyfromhigherwithinresponsepatients
levels,

in

PMNs.

of

than

PMNs

canceradults.

Onwithbacterial

contrast

the

the

hand,

effectminutes,therhG-CSF

exhibit-

on

CI,

human
effectpersons

infections

to10

enhancedpriming

elderly

otherconcentrations
forrecombinant
The

and

on

chemilumines-

in
37•Ž

untreated(rhGM-CSF).

lung(rhG-CSF)
ofsignificantly

factorpatients

factor(PMNs),

vitro.

obtained

effectSchoolcolony-stimulating
leukocytes

were

by
of

granulocyte

withfollowed

less

Kunii

Itabashi-ku,

polymorphonuclear

response,

Aoki

Kaga,

4 ng/mla

Otohiko

Masumi2-11-1,

than

ed

Ono,

on theleukocytes

Medicine,

more

responseoffactorpolymorphonuclear

Internal

Medicine,(Receivedcolony-stimulating

responseandThe

MAY 1993

wastheprimingtended

to

PMNs

of

almosteffectbefromstronghealthy

adults.

Key

words:

rhG-CSF,

rhGM-CSF,

polymorphonuclear

Introduction

Human

granulocyte

ed.

colony-stimulating

factorleukocytes,

In

CSF

this
on

chemiluminescencestudy,

thewe(G-CSF) is a naturally occurring glycoprotein with
an apparent molecular weight of 19, 600. It is amember of a family of hemopoietic growth factors

humanpolymorphonuclear

obtainedfromand regulates

macrophage

the proliferationand differentiationof

granulocyte
and macrophage
precursor cells1) A
complementary
DNA encoding human G-CSF has
recently

been cloned

coli and Chinese
scale productionand expressedin Escherichia

hamster ovary cells, and largeof recombinanthuman G-CSF(rhG-CSF)
has been established2,3). In addition toits effect on granulopoietic
cell growth and granulocyte production,

rhG-CSF

established,on the PMNsof healthy

adults has been

that

CSF).

of

We

rhG-CSFonpromisedhosts,

andelderlyCL
includingreported

with

acute

to

be

of

PMNs

patients

Materials
SubjectOureffect

PMNs
with

PMNs

in
lung

in

in

of
com-

cancerfunctionsandinfections
was(rhGM-

priming

impaired9,10),

bacterial

response

granulocyte-

of

whose

of

comparedfactorresponse

persons,

been

human

the

rhG-

(PMNs)
andstimulating

investigatedtheofresponse

adults,recombinantalso

effect

(CL)

young

colonyhadpatients

whom

the

CL

enhanced11).
andMethods

population
subjects

weremales,

7 females,in patients

23-36),

twelvediseases has not been fully elucidat-

males,

the effect on PMN functions

with underlying

with

the

leukocytes

healthyenhances a number offunctional events in mature granulocytes,
both invitro and in vivo4-8). Although the priming effect ofrhG-CSFitexaminedchemiluminescence3

females,twentymean
patients
mean

healthyage

volunteers.(1330.8+4.3
withage

years,
lung

67 .3•}10.6

range

canceryears,(9

VOL.41

range

Effect of rhG-CSF

NO.5

49-80),

infections

twelvepatients(11 males,with

1 females,meanelderlyprovidedJapan). Endotoxin

Limulusamebocyteassay

rhG-CSF

and

per-

ged

by

4.8

in the66.1•}

protein)Ltd.

(5 males,7

years,

range

68-84)

without

specific

underlying

Isolationofforhypertension

andarterioscler-

Freshlydrawn

females,

twelvekindly(Tokyo,Japan

years,

except

and

age

U/mg

chst

sons

otic

42-80)

5•~10'

bacterial

11.4

diseases

range

acute

medianage

75.2 •}

fromdisease.

Patients

with

lung

cancer:All

stagedclinicallyaccordingtheStaging

forCancer.

ClinicalLung

cer revealed
ID,

and

1 patient
9 withdiagnosis

was

mous

cell

Those

with

cluded

as

4,

our

as

infection.

The

with

a patient

with

1

with

sepsis,peritonitis,

1

and

lung

cancer,1 withcolon

cancerand

1 with

and

media

with

0.3

Co.,

Tokyo)

Co.)

weredissolvedinconcentrations

respectively.

and

coli

specifickindly(Tokyo,Ltd.

Japan)(Tokyo,human
factorcoil(moleculartoa finalsepsisMeasurement

pulus

The

identified
isolatedinused(Sigmaphosphate-buffered20 ƒÊg/ml

and

human

factor(rhG-CSF)(apparent

molecular

weightPharmaceuticalproduced
and

by

ng

of

salineasthecontrol;ofpreincuba-ofrhG-CSF,salinethemixturesandfor

20 minutes

withGermany)forThis

drug-treated
The

was

index

CL

of

at

zymosanwasmeasureda six-channelCo.,betweeneach

definedadded

continuously

Biolumat

. The

37•Ž,

was

LB

9505

chemiluminescencedrug

studied

was

as

the

and

untreated

CL

(con-

calculatedbydividing

the

PMNs

by

that

of

mean•}SD,

and

each

experi-in duplicate.

StatisticalCLwasdrug-exposed

mean

deter-

difference

PMNs.

Statistics

Co.,

Date
ment
sis

are
was

was

shown
conducted

performed

Escherichia
activityCLmixture10 minutes

non-opsonized

mined.

trol).

for

each

20 #1

After

0.9%

25 mg/

orandvolumeor(CL-index)(0.5-

rhGM-CSF)

MEM.

concentrations

index

solution

final

varioussaline

of

with

with

of

CSF

The

tion

(Berthold

ml

1 ml

to

Recombinant

specific

2-50

the

colony-stimulat-

22,000

or

to

Ltd.

Sankyo, respectively

solution.

ml

by
CL

0.1

of

adjusted

Co.,

and

concentrations

wasuntreated

Brewery

various

ml

was

Kirin

specieschemiluminescence, 0.1

protein)byandPMNs/ml(5 x 106 cells)

integralU/mg

eryth-of 1•~10'

suspension

18,8001•~108MEM

contained

Kasei

by

with

mixture(500ƒÊg)produced

thereaction

20ƒÊ1

granulocyte(0.2%)lyse

The

rhGM-CSF

Chemical

twice

for

sediment

hypotonic
toassay.

andto(Tokyowiththeoxygenluminol

wasinluminol-dependent

1 with7.4,

centri-

gradient

by

0.9%

(MEM,

follow-wasreactive

of

HEPES

40 minutes,
plasmameasured

0.1

mM

lung

sedimented

was

5 withmediumandU/ml)

subjects

PMNs

infarction,

25

tubes

salineofrhG-CSF

failure,elderly

concentration

of

of

heart

with

pellets

washedproduction

100 ngcerebral(10in MEM.

under-

ofweightadjusted

moderateorRecombinant(rhGM-CSF)

1

containing

zymosaninJapan)

Their

were

PMN

granulocyte-macrophageing(1.6%)

pneu-

Luminol

activity

providedsequentially

hypertonic

aenrginosa

pH

and

colony-stimulating
Escherichia

treated

and

Cells

blood
patients

a Ficoll-paquewererocytes.

jud-detectable

and

for

remaining

with

mild

samples.

across

2.

ileus

was

solution

The

with

were

Eagle's

g/L,

Kogyo

2

plasticex-pneumonia,

2 patientscontaining

blood

(PBS)

400•~gileus.

modifieddilute

ml,

at

in

congestive

Tokyo)

L-glutamine

fuged

30 minutes.

andleukocyte-rich

Pseudomonas

had

Three

Co.,

the

were

with

aureussputumpatients

Dulbecco's

withorganisms

ling diseases;

Chemicals

Dextranwhich

carcinoma

infection:six

sepsisfrom

Eleven

4.5%

ing

squa-

donorsin sterile

with

not

Hoe-

preparations.

infections,

can-

6,

cell

bacterial

by

PMNs

human

collected

was

rhGM-CSF

heparinizedStage

histologic

disorders

Staphylococcus

isolated

Nissuismallbacterial

causativein 3 patients.

The

follows;

abscess,
1

and

acute

was

lung

for

2 withstudy.

withsuspected,

IIIa,adenocarcinoma

inflammatory

Patients

monia.

follows:

was

was

human

healthy

cancer,International

IV cancers.

acuteclassification

was

Stage

were

stagingStagecarcinoma

from

with

with

patients

New

541

on PMNs Chemiluminescence

as

using

Student's

analy-

t-test.

Results
The

CL

responses

of PMNsin elderly

personsandCHEMOTHERAPY

542

in PMNs. About a 1.25 times increase in the inte-

the patients with lung cancer or acute bacterial
infection were investigatedand comparedwiththose of PMNs

from healthy

adults,gral value for the CL response of PMNs, comparedwith the response in untreated PMNs, was achieved
after incubating 30 ng/ml of rhG-CSF and PMNs at
3TC for 10 minutes, followed by stimulation withzymosan. Fig. 3 shows that the priming effect ofrhG-CSF on the CL response of PMNs in healthy
adults was significant beginning at a concentrationof 4 ng/ml , with the optimal concentration being 30
ng/ml , and the CL response gradually decreasing at
the higher concentrations. Fig. 4 shows the priming
effect of rhG-CSF and rhGM-CSF on the CL respo-

The CL res-ponse of PMNs in patients with bacterial infections
was greater than that of the PMNs of healthy
adults,while this response

tended to be lower thanin healthy adults in patients
elderly persons (Fig. 1).
Fig. 2 showsa typical

with lung cancer

patternandof the priming

effect of rhG-CSF on the CL response of PMNs tostimulation with non-opsonized zymosan in healthy
adults. rhG-CSF

MAY 1993

alone did not induce a CL response

Data are shown as integral CL counts for 20 minutes (means and SDs).
**Significantly different (p<0 .01) from the values in healthy adults.
CL: chemiluminescence,
Fig. 1.

CL values

PMNs: polymorphonuclear

of PMNsin elderly

persons,patients

infectioncompared

with

PMNs

(5•~105/ml)

werestimulated
with
those

incubated

with
lung

non-opsonizedcancerof healthy

withleukocytes.
zymosanand acutebacterial

adults.

rhG-CSF(30 ng/ml)ornormalsaline(control)for 10 minutes
in the presenceof luminol;
then PMNs
CL weremeasuredby stimulation
with non-opsonizedzymosan.
rhG-CSF:
recombinanthuman
granulocytecolony-stimulating
factor.

Fig. 2.

Typicalpattern

the CL response
tion

with

of the primingof PMNs

non-opsonizedeffectin a healthy
zymosan.

of rhG-CSF
adult

on

to stimula-VOL.41

Effect of rhG-CSF

NO.5

nse of PMNs

from healthy

adultswith zymosan.

rhGM-CSF

had a stronger

than rhG-CSF

at all levels

studied.

on PMNs Chemiluminescenceto stimulation
effect

The priming

effectof rhG-CSF

frompatients

Priming

effectof variousof rhG-CSF

on theCL response

healthy

adultsto stimulation

withFig. 4.
innon-opson-

effectof rhG-CSF

on the CL response

adultstoandof PMNsstimulation

with

1. Effect

Data

are

The

CL-indexof rhG-CSF
with

as

CL-index

shown

was

the

t-testfor(control),under

identical

p<0.05,Student's

elderlypersons,lungpairedof rhG-CSF.

as

data,

for
in

and

pairedpatientsof PMNs
acuteof

described

in healthy

bacterial

adults,in elderly

persons,*p<0.

**p<0.

infections

n experiments).
incomparison

incubation

t-testandcancer(mean•}SD

determined

Student's

concentrations

on the CL response

in patients

betweenMaterials
theandMethods

rhG-CSF-treated

and

05,
untreated

01,

PMNs

conditions.
•˜data,withcomparison
lungcancerbetween
and

acute

the

CL-index

bacterialinhealthy

infections,atadults,therhGM-

in healthy

non-opsonizedzymosan.

andinPriming

CSF

ized zymosan.
Table

of PMNsand from elderly

Student's
t-test for paired data, comparison
betweenrhG-CSF-treated
and rhGM-CSF-treated
PMNs
under identical incubation
conditions.
rhGM-CSF: recombinanthuman granulocytemacrophagecolony-stimulating
factor.

concentrationsof PMNson the CL responsewith lung cancer

persons was investigated and compared with that of
PMNs from healthy adults. The priming effect of

The CL-index
was determined
as describedinMaterials and Methods.
Student's
t-test for paired
data, comparison
between rhG-CSF-treatedanduntreated PMNs (control)
under identical incubation
conditions.

Fig. 3.

543

same

CHEMOTHERAPY544

MAY 1993

priming effect on the CL response than rhG-CSF.
Yuo et al. determined the production of superoxideanions induced by N-formyl-methionyl-leucylphenylanine (FMLP) using the ferricytochrome creduction test and reported that the activity of
rhGM-CSF in promoting the production of superoxide anions (priming effect) was higher than that of
rhG-CSF6'.This
is in agreement with our own
results, Although the priming effect of rhG-CSF on
PMNs from healthy adults has been studied, theeffect on PMN function in patients with underlying
diseases has been less extensively investigated. Ourinvestigation of the priming effect of rhG-CSF on
the CL response of PMNs in compromised hosts
including patients with lung cancer and elderly
Student's
1-testrhG-CSF-treatedFig. 5.

Primingof rhG-CSF
healthy

rial infectionsized

effectsof various

concentrations

on the CL response

adultspersons, whose PMNs CL responses are slightly
depressed, revealed that significant enhancement of
the CL response was achieved after PMNs werefor paired data, comparison
betweenand untreated
PMNs (control).

andpatients

to stimulation

withof PMNs
acute

with

in

bacte-

non-opson-

zymosan.

rhG-CSF in these compromised
hosts was almost
the same as in healthy adults (Table 1). The priming effect of rhG-CSF was also compared in PMNs
from healthy adults and in PMNs from patientswith bacterial

infections,in whom the CL responseof PMNs was enhanced.

Fig. 5 shows that in thesepatients a significant effect was noted starting atfrom 2 ng/ml; this was lower than the minimal level(4 ng/ml) for the priming effect in healthy adults,
the optimal level being 4 ng/ml , as opposed to 30
ng/ml in healthy adults, and the CL response
to decrease at higher levels.

tendedDiscussion
Cytokines such as G-CSF and GM-CSF have beenreported to promote in vitro PMN functions including phagocytosis,

arachidonic

acid release,andsuperoxide anion production,
in response to a stimulus4-8). In this study using a luminol-dependent
CL method , we examined

the influence of rhG-CSF

and rhGM-CSF on the potential of the PMNs ofhealthy adults to produce reactive oxygen species
by following stimulation

with non-opsonized

san . We found that rhGM-CSF

exhibitedzymo-

a stronger

preincubated with rhG-CSF at a concentration of
more than 4 ng/ml for 10 minutes, followed bystimulation with non-opsonized zymosan. Although , there have been some reports of the priming
effect of rhG-CSF on the function of PMNs obtained from compromised hosts, restoration of the
reduced superoxide production was observed instudies performed in patients with myelodysplastic
syndrome and lymphoma12.13).
However, to our knowledge, no previous investigators have reported the effect of rhG-CSF on thefunction of activated PMNs obtained from patientswith infections. Our investigation of the priming
effect of rhG-CSF on the CL response of PMNs
from patients with bacterial infections revealed
results different from those in healthy adults. In thecase of patients with infections, a significant effectwas observed starting at 2 ng/ml , which is lower
than the minimum concentration (4ng/ml) required for the priming effect in healthy adults. The
optimal level of rhG-CSF for the CL response in thepatients with infections was 4 ng/ml as opposed to
30 ng/ml in healthy adults. Although the mechanism for underlying the differences in the reactivityof rhG-CSF to the CL response of PMNs associatedwith infections is unknown, prior priming of PMNs
with rhG-CSF, which is increased in blood due toinfection") may be one explanation for the diffe-

VOL.41

Effect of rhG-CSF

NO.5

on PMNs

rence. Interaction with such cytokines as GM-CSF
tumornecrosis factor, interleukin-1 (IL-1) , and IL-8, which activate PMNs in the presence of bacterial infection, as well as interaction with bacterial
components including lipopolysaccharide (LPS) ,may also be responsible's). Furthermore, in vitro
studiesmay be required to elucidate changes that
occur in the priming effect of rhG-CSF on the CL
responseof PMNs when rhG-CSF is combined withother cytokines or LPS.
The bactericidal activity of PMNs has beenshownto be reduced in patients with lung cancer9)
andin elderly patients") and, this is thought to be acause of the high incidence of infection in suchpatients. The present study, using a CL method,
showed that rhG-CSF enhanced the function of
PMNs in producing reactive oxygen species inpathwaysgranulocyte bactericidal function.
Acknowledgments
We thank Ms. M. Ozawa and Ms. K. Ishizawa
for their technical assistance.

5)

human

Kitagawa

S,

Yuo

Y,

A,

F:

granulocytes

colony-stimulatingsuperoxide

releaseby

Biophys
Yuo

Kitagawa

S.

by

and

139:

OhsakaA,

priming

Biochem
1987

Saito
of

M,

Taka-

human

neutro-colony-stimulating

factorcolony-stimulating

Qualitative

Biochemenhances

peptide.
1143-1146,

granulocyte

M,

human

granulocytes

144:

and

Saito

factorgranulocyte-macrophagefactor:M,

humanchemotactic

CommunF: Stimulation

phils

L

Recombinant

in

the

Res
A,

ku

J Immunol

Souza

Takakustimulated

6)

granulocytes.

1987

Miura

BiophysandquantitativeRes

Commun.

differences.
171:491--497,

1990
7)

Steinbeck
by

M

8)

J,

Roth

recombinant
568,

J

A:

Neutrophil

activation

Rev

Dis

cytokines.

Infect11: 549

1989•`

MatsumotoM,

TanuraYokotalocyteMatsubara

M,

HattoriT:

Protective

S.

K,

in

effect

of

T,

H,

M,

InfectOno

humanfactormice,

MatsunoNomuracolony-stimulating

infection

granu-

on

Immun

microbial

55:2715-2720,

1987
9)

Hara

N,

ki

Ichinose

M,

69:

M:

of
in

patients

G,

Lanzio1682-1687,

G,

activity32:308-316,

M,

12)

Barbour

A G,

Allredinhealthy

from

C D,

patients

InfectYuo

A,

Kitagawa

Y,

Itoh

141:
S,

S,

C 0,

active

N,
of

human

trationofrecombinant-stimulatingT,lymphoma.

Blood
M,

S,

A,

Koma-

F,

chronic

404•`411,

Saito

Miura

Y,

In

vivo

acti-

M:

by

Hirai

M,

Kurosawa

S,

M:

Levelsof

serum

granulocyte

ing

factorinpatientswithadminis-granulocyte
with74: 2743-2748,

H,

1987

S,

functions

H,

with

and

patients

thepatients

70:

human
in

human
repairs

Sakamoto

Tsutsumi

1990

infec-

Urabe

inneutrophil

Kawakami

H R:
leuko-

bacterial

BloodfactorHill

factorTakakuvationGer-

F: Recombinant

Kitagawa

Takanashi

pha-

1980

Okabe

leukemia.
A,onvitro:

Solberg

syndromes

myelogenous

S,

age

subjects.

neutrophils

myelodysplastic

1962-1964,

of

human

colony-stimulatingof

Romanoin

14-26,

Takakuabnormalities

14)

T,

In)uencepolymorphonuclear

with

Dis

tsu

OhsakaandCancer1986

bygranulocyte

13)

AnfossiG:

Chemiluminescence

J

Kawasa-cancer.

leukocytes

ontology

tion.

T,

leukocytes
lung

Calcamuggi

gocytic

cytes

Yano

anion-generating

withpolymorphonuclear

11)

H,

1992

Emanuelli
AnfossiAsoh

Superoxide

polymorphonuclear

monocytes

10)

Y,

Ohta

activity

References

1) Metcalf D: The molecular biology and functionsof the granulocyte-macrophagecolony-stimulating factors. Blood 67: 257-267, 1986
2) Souza L M, Boone, T C, Gabrilove J, Lai P H,
Zsebo K M, Murdock DC. Chazin V R, Bruszewski J, Lu H, Chen K K, Barendt J, Platzer E,
Moore M A S, Mertelsmann R, Welte K: Recombinant human granulocyte colony-stimulating
factor: Effects on normal and leukemic myeloid
cells. Science 232: 61-65, 1986
3) Nagata S, Tsuchiya M, Asono S, Kaziro Y,
Yamazaki T, Yamamoto 0, Hirata Y, Kubota
N, Oheda M, Nomura H, Ono M: Molecularcloning and expression of cDNA for humangranulocyte colony-stimulating
factor. Nature
319:415-417, 1986
4) Sullivan R, Griffin J D, Simons E R, Schafer A I,
Meshulam T, Fredette J P, Maas A K, Gadenne
A S, Leavitt J L, Melnick D A: Effects of recombinant human granulocyte and macrophage colony-stimulating
factors on signal transductionin34223430,

patients with lung cancer and elderly persons.
Hence, the clinical employment of rhG-CSF may beof value as adjuvant therapy in the treatment andprevention of serious infections in these patients
when granulocytopenia is present, not only to
inducegranulocyte proliferation but also to activate

545

Chemiluminescencecolonymalignant

1989
Kumakawa

Mori

M,

T,

Abe

Fukushimacolony-stimulatinfections.

Blood

76:

CHEMOTHERAPY

546

15)

Bone

R C: The

pathogenesisofsepsis.

AnnalsofInternal

MAY 1993

Medicine,

115:457•`469,

1991

遺 伝 子組 み換 え型 ヒ ト顆 粒 球 コ ロニ ー刺 激 因子 のヒ ト好 中球 機能 にお よぼ す効 果

大谷津

功 ・斧

康 雄 ・青 木 ます み 。国 井
帝京大学医学部第二 内科'

遺 伝 子 紹 み 換 え型 ヒ ト顆 粒 球 コ ロ ニ ー 刺 激 因 子(rhG‑CSF)の
効 果 をchemiluminescence(CL)法得 た 好 中 球 と37℃ で10分
CSF4ng/ml以
rhG‑CSFのCL反を 用 い てinvitroで

間 保 温 し た 後 に,非ヒ ト好 中 球 機 能 に お よ ぼ す

検 討 し た。rhG‑CSFをオ プ ソ ニ ン 化zymosanで

健康 成人 よ り

刺 激 す る と,rhG‑

上 の 濃 度 で 好 中球 のCL反応 が 未 処 理 の も の に 比 較 し て 有 意 に 増 強 した 。 この応 に お よぼ すpriming効

果 は遺 伝 子 組 み換 え型 ヒ ト顆 粒 球 ・マ ク ロ フ ァ ー ジ

ー コ ロ ニ ー 刺 激 因 子(rhGM‑CSF)に

比 較 し て弱 か っ た 。rhG‑CSFは中 球 に 対 し て も健 康 成 人 の 場 合 とほ ぼ 同 等 のpriming効の 活 性 化 し た 好 中 球 に 対 す るpriming効濃 度 で 弱 い傾 向 が み られ た 。
*東 京 都 板 橋 区 加 賀2‑114

乙彦

果 は,健

肺 癌 患 者 や 高 齢 者 の好果 を 認 め た 。 一 方,細菌 感 染症 患 者

康 成 人 の 場 合 と 異 な り,低 濃 度 で 強 く,高