信州医誌，58⑵：57〜68，2010

Irradiation Stimulates Human Lung Fibroblasts to Release
Inﬂammatory Cell Chemotactic Activity
Makoto KURAI ＊ and Sekiya KOYAMA
1） Department of Surgery (II), Shinshu University School of Medicine
2） Department of Pulmonary Internal Medicine,National Hospital Organization Chushin Matsumoto Hospital

Background :Radiation therapy plays an important role in the treatment of lung cancer. However, it alsoradiation therapyoften induces serious complications such as radiation‑induced pneumonitis and its underlying
mechanism remains to be elucidated.
Objectives:The present study examined whether radiation might stimulate human lung ﬁbroblasts (HLF)torelease neutrophil and monocyte chemotactic activity (NCA and M CA).
Methods:HLF were exposed to varying doses of radiation (3‑12 Gy)at varying incubation times (12‑72 hr).
The chemotaxis assays of NCA and M CA were performed by a 48‑well microchemotaxis chamber method.
The eﬀect of polyclonal antibodies of various cytokines on NCA and MCA was evaluated.Furthermore,theprotein and mRNA expression of these eﬀective cytokines was assessed by ELISA and RT‑PCR.
Results:HLF released NCA and MCA in response to irradiation in a dose‑and time‑dependent manner.NCA
was signiﬁcantly inhibited by anti‑interleukin (IL)‑8 antibody and M CA was signiﬁcantly attenuated by anti‑
monocyte chemoattractant protein (MCP)‑1 antibody. The protein secretion of IL‑8 and MCP‑1 wassigniﬁcantly increased by irradiation,and mRNA expression of IL‑8 and MCP‑1 was upregulated by irradiation.
Conclusion :These ﬁndings suggest that HLF may,at least partly,participate in the development of radiation‑induced pneumonitis. Shinshu Med J 58 : 57 ―68, 2010
(Received for publication November 26, 2009 ;accepted in revised form January 8, 2010)
Key words:irradiation, human lung ﬁbroblast, chemotaxis, IL‑8, MCP‑1

ductive cough,fever and dyspnea on eﬀort,develops

Introduction

2 to 12 weeks after completion of a course of radio-

Radiation therapy plays an important role in thetreatment of localized lung cancer

therapy. It is thought that the early onset of. However, it

symptoms is indicative of a more serious andoften induces serious complications,including radia-

protracted clinical course . However, the under-tion‑induced pneumonitis and ﬁbrosis.In particular,

lying molecular and cellular mechanisms of radia-

radiation‑induced pneumonitis occurs in 5 to 20%

tion‑induced pneumonitis remain to be elucidated.

of patients irradiated for lung cancer and is some-

Pathological characteristics of radiation‑induced

times a limiting factor in radiation therapy. An

pneumonitis are interstitial edema, interstitial andearly acute pneumonitis, characterized by non‑pro-alveolar cellular inﬂammation, and an increase in
the number of type II pneumocytes .An increase in

Corresponding author:Makoto Kurai
Department of Surgery (II),Shinshu University School ofinﬂammatory cells including

neutrophils andMedicine, 3‑1‑1, Asahi, Matsumoto‑city,

monocytes is often found in the lumen of airways,

Nagano‑prefecture, 390‑8621, Japanthe interstitium and the alveolar spaces in rat fol-

No. 2, 201057

Kurai・Koyama

lowing thoracic irradiation . Therefore, it is

growth factor, suggesting their contribution to cer-thought that inﬂammatory cells may play an impor-tain disease states

tant role in radiation‑induced pneumonitis. Seques-that bleomycin and cyclophosphamide stimulate

tration of peripheral blood neutrophils andhuman lung ﬁbroblast (HLF) to release NCA andmonocytes within the lung is characteristic of a

M CA, including IL‑8, granulocyte colony‑stimulat-

number of acute and chronic pulmonary dis-ing factor (G‑CSF), MCP‑1, and GM ‑CSF

eases

.The presence of neutrophils is determined

Therefore the ﬁbroblast, because of its anatomic

by the local generation of chemotactic agents,

location,is in a pivotal position to participate in andwhich direct neutrophil migration from the vascularestablish direct communication between interstitialcompartment to the alveolar space along

and vascular events in pulmonary inﬂammation andchemotactic gradients.The alveolar macrophage isﬁbrosis.

. Recently, it was reported.

also derived predominantly from diﬀerentiatedAlthough airway epithelial cells and alveolar

peripheral blood monocyte migration and to a lim-

macrophages may play a role in inﬂammatory cell

ited extent from local macrophage replication .

migration from the interstitium to the alveolar andAlthough elicited neutrophils and macrophages

bronchial spaces in response to irradiation,the role

serve a vital role in host defense against a number

of HLF in inﬂammatory cell recruitment inof organisms,the presence of increased numbers of

response to irradiation is uncertain.The purpose ofactivated inﬂammatory cells can lead to tissue

the present study was to determine whether HLF

injury and ﬁbrosis‑promoting activities via the

could participate inexcessive elaboration of inﬂammatory cytokines,

inﬂammatory cells into the lung. Speciﬁcally, theproteolytic enzymes, and oxygen radicals. Sub-

possibility of lung ﬁbroblasts releasing NCA andstantial investigation has focused on the alveolar

M CA in response to irradiation was evaluated.The

macrophages as a primary source of the chemo-

results demonstrated that HLF released NCA andtacticfactor .However,neutrophilchemotacticactivity

M CA in response to irradiation,including IL‑8 and(NCA)

M CP‑1,suggesting that HLF may participate in theandmonocyte

chemotactic

activity(M CA)have been found to beproduced byendothelial
cells , lung ﬁbroblasts , and pulmonary epithelial
cells .
The ﬁbroblast is the principal cell of most connec-

the recruitmentofdevelopment of radiation‑induced pneumonitis.
M aterials and Methods
Culture of HLF

tive tissues and is involved in constituting col-We used fetal HLF (lung,diploid,human,passage

lagenous and noncollagenus components of the29) which are an established cell line and commer-

extracellular matrix. This synthetic activity serves

cially available (American Type Tissue Culture

an important structural function by providing a

Collection,Manassas,VA).The HLF were suspend-

frame work for organ integrity.In additional to thised at 1.0x 10 cells/ml in Hamʼs F‑12 medium

traditionally accepted function, recent studies

supplemented with penicillin (50 U/ml,GIBCO BRL

have demonstrated that ﬁbroblasts not onlyserve tolife technologies, Grand Island, NY), streptomycin

maintain the connective tissue but are important(50μg/ml, GIBCO BRL), and fungizone (2μg/ml,

participants in the orchestration of acute and

GIBCO BRL) and 10% heat‑inactivated fetal

chronic inﬂammation. In this context, ﬁbroblasts

bovine serum (GIBCO BRL).After the cells reachedreleased monocyte chemoattractant protein (MCP)‑

conﬂuence,they were harvested with 0.25% trypsin

1, granulocyte‑macrophage colony‑stimulating fac-

and 0.1% etylenediamine tetraacetic acid in phos-tor (GM ‑CSF), transforming growth factor (TGF)‑

phate buﬀered solution (Sigma, St. Louis, M O),

β in response to interleukin (IL)‑1 β and tumor

centrifuged at low speed (250 x g, 5 min), and were

necrosis factor (TNF)‑α, and platelet‑derived

re‑suspended in fresh medium at 1x10 cells/ml in58

信州医誌 Vol. 58

Lung ﬁbroblast stimulation by irradiation

30 mm tissue culture dishes. The cells reachedconcentration of 5.0X10 cells/ml. This suspension

conﬂuence after 4‑6 days incubation,and were thenwas used for the monocyte chemotaxis assay.

used for the experiments.
Exposure of HLF to irradiation

The chemotaxis assay was performed by a 48‑
well microchemotaxis chamber (NeuroProbe Inc.,

The medium was removed from cells by washing

Cabin John, MD), as described previously . The

twice with serum‑free Hamʼs F‑12, and cells were

bottom wells of the chamber were ﬁlled with 25μl

incubated in the presence and absence of irradia-

of ﬂuid containing the chemotactic stimulus ortion. To determine the dose‑ and time‑dependent

media in duplicate. A 10μm thick polyvinylpyr-

release of NCA and MCA the cells were exposed to

rolidone‑free polycarbonate ﬁlter (Nucleopore,varying doses of radiation (3,6,9,12 Gy,by high X‑

Pleasanton, CA), with a pore size of 3μm forray source,1.5 M eV,linear accelerator,Mitsubishi,

neutrophil chemotaxis and 5μm for monocyte

Tokyo, Japan), and were incubated for 12, 24, 48,

chemotaxis,was placed over the bottom wells.The

and 72 hr at 37 ℃ in a humidiﬁed 5% CO atmo-

silicon gasket and upper pieces of the chamber were

sphere. Irradiation did not cause injury (no deform-

attached,and 50μl of the cell suspension was placed

ity of cell shape,no detachment from tissue culture

into the upper wells above the ﬁlter.The chambers

dish, and greater than 98% of cells were viable bywere incubated in humidiﬁed air in 5% CO at 37 ℃

trypan blue exclusion) after 48 hr incubation at 6for 30 min for neutrophil chemotaxis and 90 min for

Gy.The supernatant ﬂuids were then harvested andmonocyte chemotaxis. After incubation the cham-stored at ‑80 ℃ until assayed.At least six separate

ber was disassembled and non‑migrated cells were

HLF supernatant ﬂuids were harvested for each

wiped away from the ﬁlter. The ﬁlter was thenexperimental condition.

immersed in methanol for 5 min, stained with Diﬀ‑

M easurement of NCA and MCA

Quik (American Scientiﬁc Product, McGaw Park,

Polymorphonuclear leukocytes were puriﬁed

IL), and mounted on a glass slide. Cells completelyfrom heparinized normal human blood by the

migrated through the ﬁlter were counted by using

method of Boyum . The resulting cell pellet, as

light microscopy in ten random high power ﬁelds

determined by trypan blue and erythrosine exclu-(HPF, ×1000)per well.

sion, consisted of ＞96% neutrophils and ＞98%

To ensure that monocytes, but not lymphocytes,

viable cells. The cells were suspended in Geyʼswere the primary cells that migrated in thebalanced salt solution (GIBCO BRL)containing 2%

monocyte chemotaxis assay,some membranes were

bovine serum albumin (BSA, Sigma) at pH 7.2 tostained with alpha‑naphthyl acetate esterase

give a ﬁnal concentration of 3.0x10 cells/ml. Thisaccording to the manufacturerʼ
s directions (Sigma).

suspension was used for the neutrophil chemotaxis

All volunteers provided written informed consent

assay.
M ononuclear cells for the chemotaxis assay wereobtained from normal human volunteers by Ficoll‑in advance for the use of their bloods. The study
was performed in compliance with the Declarationof Helsinki.

Hypaque density centrifugation to separate red

Eﬀects of LTB4 receptor antagonist on NCA

blood cells and neutrophils from mononuclear cells.

and MCA released from HLF in response to

The preparation routinely consisted of 30% large

irradiation

monocytes and 70% small lymphocytes determined

LTB receptor antagonist (ONO 4057, Ono Phar-by morphology and alpha‑naphthyl acetate esterase

maceutical Co.,Tokyo,Japan)at a concentration ofstaining (Sigma) with ＞98% viability as assessed10by trypan blue and erythrosin exclusion. The cells

LTB as NCA and M CA in the supernatant ﬂuids

were suspended in Geyʼs balanced salt solution

incubated with irradiation at 6 Gy for 48 hr.

M was used to evaluate the involvement ofcontaining 2% BSA (Sigma)at pH 7.2 to give a ﬁnal
No. 2, 201059

Kurai・Koyama

Eﬀects of polyclonal antibodies to IL‑8, G‑

Japan)and then ampliﬁed for 26 cycles in PERKIN

CSF,GM ‑CSF,MCP‑1,M ‑CSF,or RANTES on

ELMER Gene Amp PCR System 9600‑R (denatura-

NCA and MCA released from HLF in responsetion at 94 ℃ for 30 sec,primer annealing at 55 ℃ for

to irradiation

30 sec,and primer extension at 72 ℃ for 30 sec).TheThe neutralizing antibodies to IL‑8,G‑CSF,MCP‑

sequences of IL‑8 and MCP‑1 used in the present

1,GM ‑CSF,M ‑CSF,and RANTES were purchased

study were as follows:from Genzyme (Cambridge, M A). These antibodies‑AAC ATG ACT TCC AAG CTG GC‑
IL‑8 sense,5ʼ

were obtained from mouse and added to HLF super-‑ACT GGC ATC TTC ACT
3ʼ
, and anti‑sense, 5ʼnatant ﬂuids,which were harvested at 48 hr incuba-‑CAGCCAGATGGAT TC‑3ʼ
; MCP‑1 sense, 5ʼtion in response to 6 Gy irradiation at the suggested‑GTG GTCCAATCAATGC‑3ʼand anti‑sense, 5ʼ

concentrations to inhibit the migratory potential of‑
CAT GGA ATC CTG AA‑3ʼ
; β‑actin sense, 5ʼthese cytokines. Then these samples were used for

TGA CCC AGA TCA TGT TTG AG‑3ʼand anti‑

chemotactic assay. The antibodies inhibited each

‑TCA TGA GGT AGT CAG TCA GG‑3ʼ
sense, 5ʼ

chemokine ‑induced neutrophil and monocyte

Preliminary studies indicated that more than 26

chemotactic activity, and each antibody did notcycles were subsaturating for the mRNA tested,inﬂuence the neutrophil and monocyte chemotaxis

and thus was appropriate for comparison of relative

induced by activated serum or fM LP (formyl meth-levels of mRNA between groups. PCR products

ionyl leucyl phenylalanin) , as we have previously

were separated by electrophoresis on 3% agarose.We used non‑immune IgG as negativegel. β‑actin mRNA, which has been shown not toreported
control.

M easurement of IL‑8 and M CP‑1 proteins inchange by stimulation, was measured in both normal and stimulated RNA samples at each point,

HLF supernatant ﬂuids

using the same cDNA that was analyzed for cyto-

Because IL‑8 and M CP‑1 antibodies signiﬁcantly

kines. The intensity of the bands was quantiﬁed byinhibited NCA and M CA in the supernatant ﬂuids,

densitometry. The results were expressed as thethe concentrations of IL‑8 and MCP‑1 in HLF

ratio of intensity to the β‑actin.

supernatant ﬂuids cultured for 48 hr after 6 Gy

Statistics

irradiation were measured by enzyme linked im-

In experiments where multiple experiments weremunosorbent assay(ELISA)according to the manu-

made, diﬀerences between groups were tested forfacturersʼdirection. IL‑8 and MCP‑1 kits weresigniﬁcance using one‑way analysis of variancepurchased from R&D systems (M inneapolis, M N),

with Fisherʼs multiple range test applied to data at

and the minimum detectable concentration was 10.0

speciﬁc time and dose points.In experiments where

pg/ml for IL‑8 and 31.3 pg/ml for MCP‑1.

a single measurement was made, the diﬀerences

Evaluation of IL‑8 and M CP‑1 mRNA expres-between groups were tested for signiﬁcance using

sions

Studentʼs paired t‑test. In all cases, a p value less

Reverse transcriptase‑polymerase chain reaction

than 0.05 was considered signiﬁcant.Data in ﬁgures(RT‑PCR)was used to evaluate mRNA expressionof IL‑8 and M CP‑1 in HLF cultured for 48 hr after
6 Gy irradiation.Total cellular RNA was extracted
from adherent cells using a modiﬁcation of theand tables were expressed as means±S.E.
Results
Release of NCA and MCA from HLF

methods of Chomczynski and Sacchi . One micro-

HLF released NCA and MCA in a dose‑depen-gram of total RNA was reverse‑transcribed into

dent manner in response to irradiation (Fig.1A,B).

complementary DNA (cDNA) using cDNA synthe-

The lowest does of irradiation to stimulate HLF

sis kit (MuLV Transcriptase, Rnase Inhibitor insigniﬁcantly was 3 Gy for NCA and MCA. Increas-

PERKIN ELM ER,Dnase I in TAKARA Co.Tokyo,

ing doses of irradiation progressively increased the60

信州医誌 Vol. 58

Lung ﬁbroblast stimulation by irradiation

release of NCA and MCA up to 12 Gy. Less than 3

response to irradiation in a time‑dependent manner

Gyirradiation did not signiﬁcantlyrelease NCA and(Fig. 2A, B). The release of NCA and MCA was

MCA from human lung ﬁbroblast.

signiﬁcantly increased after 12 hr exposure to irra-

Although HLF released NCA and MCA constitutively,HLF further released NCA and M CA in

diation (Fig. 2A, B). The release of chemotactic
activity reached a plateau after 48 hr.

Fig.1 Dose‑dependent release of neutrophil (panel A) and monocyte chemotactic activity (panel B) in
response to irradiation from HLF monolayers after 48 hr incubation (n =12 monolayers).
Values are expressed as means±S.E.
p＜0.05 compared with supernatant ﬂuids without irradiation.

Fig.2 Time‑related release ofneutrophil(panel A)and monocyte chemotactic activity(panel B)in response
to 6 Gy irradiation (open circles) and baseline release (closed circles) from HLF monolayers (n =12monolayers).
Values are expressed as means±S.E.
:p＜0.05 compared with medium alone.
#:p＜0.05 compared with supernatant ﬂuid without irradiation.
No. 2, 2010

61

Kurai・Koyama

Conﬁrmation that the migrated cells were

NCA, but not signiﬁcantly. NCA in the crude sam-monocytes was provided by the following lines ofples was slightly attenuated by the addition ofevidence:1) ＞90% of the migrated cells appeared

LTB4 receptor antagonist, but this too was not

to be monocytes morphologically by light micros-signiﬁcant (Fig. 3A). The combination of anti‑IL‑8

copy;2) ＞90% of the migrated cells were esterase

and G‑CSF antibodies, and LTB4 receptor antago-

positive; and 3) lymphocytes puriﬁed by allowing

nist inhibited NCA completely (p＜0.05, Fig. 3A).

the monocytes to attach to plastic and tested in theAnti‑MCP‑1 antibodies inhibited M CA (p＜0.05,

chemotaxis assayyielded 0‑20% of the chemotactic

Fig. 3B). In contrast, Anti‑GM ‑CSF, M ‑CSF andactivity of the monocyte preparation.

RANTES antibodies did not inhibit M CA (Fig.3B).

Inhibition of NCA and M CA by polyclonal

M CA in the crude samples was not inhibited by the

antibodies to IL‑8,G‑CSF,M CP‑1,GM ‑CSF,M ‑addition of LTB4 receptor antagonist (Fig.3B).The

CSF, and RANTES, and by LTB4 receptor

combination of anti‑MCP‑1,GM ‑CSF,M ‑CSF andantagonist

RANTES antibodies, and LTB4 receptor antago-

Because HLF had the potential to release chemo-

nist also similarly inhibited MCA completely (p＜0.

kines, and the chemokines released from HLF

05, Fig. 3B).

might be responsible for NCA and M CA, we used

Non‑immune IgG was used to evaluate the eﬀect

polyclonal blocking antibodies to IL‑8, G‑CSF,

of nonspeciﬁc antibody. Non‑immune IgG did not

MCP‑1, GM ‑CSF, M ‑CSF and RANTES. Amongattenuate the NCA and MCA in the same irradia-

these antibodies,anti IL‑8 antibodies inhibited NCAtion‑conditioned medium.

(p＜0.05, Fig. 3A). Anti G‑CSF antibody attenuatedFig.3 Panel A :Inhibition of NCA in HLF supernatant ﬂuids harvested at 48 hr in response to 6 Gy ofirradiation combined with anti‑IL‑8 antibody, anti‑G‑CSF antibody, or LTB4 receptor antagonist(n =12 monolayers).
Panel B :Inhibition of MCA in HLF supernatant ﬂuids harvested at 48 hr in response to 6 Gy ofirradiation combined with anti‑MCP‑1 antibody,anti‑GM ‑CSF antibody,anti‑M ‑CSF antibody,anti‑
RANTES antibody, or LTB4 receptor antagonist (n =12 monolayers).
Chemotactic activity is on the ordinate, and the experimental groups are on the abscissa.
Values are expressed as means±S.E.
:p＜0.05 compared with untreated supernatant ﬂuids.
62

信州医誌 Vol. 58

Lung ﬁbroblast stimulation by irradiation

Fig.4 The release ofcytokines from HLF in response to irradiation.Panel A shows the release ofIL‑8 from
HLF in response to irradiation at 6 Gy for 48hr. Panel B shows the release of MCP‑1 in response toirradiation from HLF.
Values are expressed as means±S.E.
p＜0.05 compared with supernatant ﬂuids without irradiation.

Fig.5 Augmentation ofIL‑8 and MCP‑1 mRNA expression by6 Gyirradiation after 48 hr incubationin HLF monolayers. Data presented is representative of three experiments. The densitometry
data with IL‑8 is expressed as a ratio of IL‑8 mRNA to β‑actin mRNA (IL‑8 mRNA/ β‑actinmRNA).The densitometry data with M CP‑1 is expressed as a ratio of MCP‑1 mRNA to β‑actinmRNA (M CP‑1 mRNA/ β‑actin mRNA).

Release of IL‑8 and M CP‑1 from HLF

Upregulation of IL‑8 and MCP‑1 mRNA

HLF was exposed to 6 Gy radiation and incubat-

expression from HLF in response to irradiation

ed for 48 hr after irradiation. The concentration of

HLF was exposed to 6 Gy radiation and incubat

IL‑8 and M CP‑1 in the culture supernatant ﬂuid

ed for 48 hr after irradiation.The expression of IL‑

signiﬁcantly increased (p＜0.05, Fig. 4A, B).

8 and M CP‑1 mRNA was upregulated (Fig. 5).

No. 2, 2010

63

Kurai・Koyama

addition, one of the pathological ﬁndings of radia-

Discussion

tion‑induced pneumonitis in rat following thoracic

In the present study, HLF released NCA andirradiation was inﬁltration of inﬂammatory cells,

MCA by irradiation in a dose‑ and time‑dependent

including neutrophils and monocytes,into the inter-manner. Pretreatment by anti‑IL‑8 antibody inhib

stitium and alveolar spaceited NCA released from HLF.Pretreatment byanti‑

reports, hypersensitive immune reaction mediated

MCP‑1 antibody inhibited MCA released fromby leukocytes is thought to be closely involved in

HLF.In response to irradiation,the release of IL‑8

the radiation‑induced lung injury

and MCP‑1 was signiﬁcantly increased. Further-sensitive immune reaction that recruits and acti-

more, the mRNA expression of IL‑8 and M CP‑1

vates large numbers of leukocytes often involves

was upregulated by irradiation.These data suggest

speciﬁc chemotactic mediators . In this context,that an interaction between HLF and irradiation

previous studies have shown that dermal andmay modulate inﬂammatory cell recruitment fromsynovial ﬁbroblasts can release soluble chemotactic

the vasculature to the interstitium after irradiation

factors that direct the migration of neutrophils andthrough the generation of chemotactic cytokines.

monocytes in response to TNF and IL‑1

. Based on these. Thus hyper-. The

Although the pathogenesis of radiation‑induced

present study demonstrated that HLF releasedpnuemonitis is complex and not yet fullyelucidated,

NCA and M CA constitutively and further in

it is thought that part of the pathogenesis of radia-

response to irradiation. Thus, lung ﬁbroblasts maytion pneumonitis is concerned with free radicals .

modulate their local immunologic environment by

The ionizing radiation collides with and excites

releasing chemotactic activity for both neutrophils

electrons, which in turn generate free radicals .

and monocytes, and may contribute to the lungFree radicals are highly reactive molecules capableinﬂammation in addition to other uncertain reasons.

of causing the scission of covalent bonds, resulting

In animals,a number of authors have described ain molecular changes that can lead to signiﬁcantthreshold dose level before any biochemical orbiochemical, structural, and functional abnormal-

histological changes begin to occur

ities. Damage to structural molecules can lead tofollowed by a steep sigmoid dose‑response curve

immediate eﬀects,such as leaky cell membranes orwith increasing lethality with increasing dose.This

faulty transport of intracellular material, and thenhas been documented by Travis and Tucker in a

resulting in rapid cell death.Moreover,free radicals

review of a number of experiments of single‑doseare also capable of causing direct damage to DNA,lung irradiation in animals .The studybyVan Dyk

which would lead to cell death.Although there is noof humans treated by single‑dose whole‑lung irradi-

doubt that direct damage to cells by irradiation isation shows a similar threshold level and steep

one etiology of radiation‑induced pneumonitis,radi-

sigmoid dose‑response curve with increase in pul-

ation ‑induced pneumonitis sometimes extends

monary symptoms and lethality with increasing

beyond the irradiated lung ﬁeld and tends to follow

dose . The probability of pneumonitis increased

a rapid course which is severe and fatal .In such afrom zero at single fraction of less than 7.5 Gy, tocase, radiation‑induced lung injury cannot be ex-

5% at 8.2 Gy, to 50% at 9.3 Gy, and 95% at 10.6

plained solely by the direct action of radiation.It is

Gy . This has also been documented by Mah from

reported that regional increases of lymphocytes inthe same institution in a re‑evaluation of fractionat-

irradiated lung have been revealed in bron-

ed irradiation converted to estimated single dose,

choalveolar lavage ﬂuid (BALF)

and his-again showing a steep sigmoid dose‑response

tological examinations .However,it is also report-

curve . Thus, lung damage from radiation can

ed that the ﬁndings were similar between theoccur immediately after the start of treatment andirradiated lung and the “
out of ﬁeld”lungis dose‑dependent. These studies document a dose‑

64

. In

, which is

信州医誌 Vol. 58

Lung ﬁbroblast stimulation by irradiation

related pulmonary toxicity. The study by Johnson

recruiting neutrpohils at the local microenviron-et al shows that the level of mRNA of TGF‑βment in response to irradiation. Furthermore,increased from 5 Gy in C57BL/6 mice .Rube et al

although the blocking antibody to anti‑G‑CSF anti-

show that after exposure to a single radiation dosebody or LTB4 receptor antagonist alone did notof 6 Gy in C57BL/6 mice, the lung tissue revealed

attenuate NCA signiﬁcantly,the combination of G‑

only a minor radiation‑mediated TGF‑beta mRNACSF antibody and LTB4 receptor antagonist withresponse .But onlya minor histopathologic changeanti‑IL‑8 antibody further inhibited NCA moreis seen after thoracic irradiation with 6 Gy. His-

strongly than anti‑IL‑8 antibody alone. Therefore,

tological change therefore seemed to be seen over

G‑CSF and LTB4 may have the potential to be

10 Gy,but an increase in cytokines and their mRNA

involved in NCA by irradiation.

levels seemed to be seen from 5 Gy in the animal

It has been reported that MCP‑1 was detected inmodel.Furthermore,Brach et al document that thelung tissue or BALF after irradiation in the animallevel of NF KB binding activity to DNA is in-modelcreased from 2 Gy in human KG‑1 myeloid leuke-lung ﬁbroblasts released MCA in response to irradi-

mia cell lines .Our study shows that the release ofation, some have documented that lung ﬁbroblasts

NCA and M CA from HLF is increased from ahave the potential to release MCA,such as MCP‑1,

single 3 Gy irradiation. Based on the above and

GM ‑CSF, RANTES, etc. in response to LPS andpresent reports,a verylow dose of single irradiation

bleomycin , cyclophoshamide , TNF, and IL‑1 .

could cause cellular response without histologic

Our studyshows that the blocking antibodyto MCP‑

changes.

1 signiﬁcantly attenuated M CA from HLF and. Although there were no reports that

Recent research has supported the view that a

conﬁrmed the release and production of M CP‑1

variety of cytokines play important roles duringfrom HLF. Thus HLF released MCP‑1, at least

radiation‑induced lung injury.Some reports suggest

partly, as the responsible M CA.

that cytokines such as IL‑6,IL‑1,TNF‑α,and TGF‑

Our results demonstrated that irradiation

βhave the potential to be a predictor of radiation

stimulated HLF to induce mRNA expression of IL‑

pneumonitis, while Hart et al have reported

8 and M CP‑1. However, the exact mechanism forthat blood IL‑8 level has a predictive value forirradiation to stimulate ﬁbroblasts resulting in thesymptomatic radiation‑induced pneumonitis inexpression of IL‑8 and MCP‑1mRNA and thepatients receiving thoracic radiation . Althoughrelease of cytokines is uncertain. There is now

resident tissue macrophages are recognaized as theevidence that irradiation may induce synthesis ofmajor local cellular source of chemokines, someinﬂammatory gene products . Gene transcription,

reports have suggested that lung ﬁbroblasts havean important prerequisite of protein synthesis, isthe potential to release NCA, such as IL‑8 and G‑

under the control of a group of proteins known as

CSF activities constitutivelyand further in response

promotors or transcriptional factors, such as AP‑1

to LPS and bleomycin ,cyclophosphamide ,TNF,

response elements and NFkB. These factors are

and IL‑1 .Furthermore Daniele Brouty‑Boye et alimportant inducers of transcription of many genes,

reported that the transcription for IL‑8 cytokine

especially cytokine genes.Irradiation stimulates thewas detected in cultured ﬁbroblasts isolated from asynthesis and/or binding to DNA of these factors .

variety of human tissues and pathogenic states,

If the synthesis and activation of these transcription

including radiation ﬁbrosis . Our study shows thatfactors is indeed directlyinduced byirradiation,it is

the blocking antibodyto IL‑8 attenuated NCA fromquite likely that transcription of a wide variety of

HLF signiﬁcantly and moreover IL‑8 was detected.

AP‑1, and NFkB responsive genes will result.

These ﬁndings suggest that IL‑8 may play a key

These ﬁndings might be involved in the expressionrole in the pathogenesis of pulmonary disorders byof mRNA of IL‑8 and M CP‑1 by irradiation.

No. 2, 2010

65

Kurai・Koyama

Because many stimuli, including IL‑1 beta, TNF

release of IL‑8 and MCP‑1 from HLF was inalpha, and LPS caused similar nuclear reactions,creased byirradiation.The mRNA expression ofIL‑

the lung infection observed in clinical settings may

8 and MCP‑1 in HLF was upregulated by irradia-

augment these responses.

tion. These data suggest that HLF might partici-

In conclusion, irradiation stimulated HLF topate in the development of radiation‑induced

release NCA and M CA. The released activity was

pneumonitis by the release of NCA and MCA inblocked by anti‑IL‑8 and MCP‑1 antibodies. The

response to radiation exposure.

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