Brucella abortus感染による流産におけるRANTESの関与

誌名誌名 The journal of veterinary medical science

ISSNISSN 09167250

巻/号巻/号 707

掲載ページ掲載ページ p. 681-686

発行年月発行年月 2008年7月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

FULL PAPER Bacteriology

Regulated upon Activation Normal T-Cell Expressed and Secreted (RANTES)

Contributes to Abortion Caused by Brucella abortus Infection in Pregnant乱1ice

Kenta W ATANABE1)， Natsumi 1W A11

)， Masato T ACHIBANA 1)， Hidefumi FURUOKA2)， Hiroshi SUZUKI3) and

Masahisa WATARA11)*

I)Departments of Applied Veterinary Science， 2Jpathological Science and 3JResearch Center for Protozoan Diseases， Obihiro Uniνersity of Agriculture and Veterinary Medicine， Obihiro， Hokkaido 080-8555， Japan

(Recei ved 17 0巴cember2007/Accepted 25 March 2008)

ABSTRACT. 8rucella abortus (8. abortus) is a facultative intracellular pathogen that can survive inside macrophages and trophoblast giant C巴lls，and the causative agent of brucellosis. [n the present study， we found that production of regulated upon activation normal T-cell expressed and secreted (RANTES) due to 8. abortus infection contributes to abortion in pregnant mice. 8. abortus infected pregnant interferon-y (TFN-y) knockout mice di巴dwithin 15 days of inf，巴ction，but non-pregnant [FN-y knockout mice were still alive. With infec-tion by wild type 8. abortus， a larg巴 amountof RANTES production was observed in pregnant IFN-y knockout mice， and induction of RANTES was also observed in normal pregnant mice infected with the wild typ巴， but not in those infected with the intracellular repli-cation-defective mutant. Production of RANTES and IFN-y were inhibited in mice inoculated with the respective RANTES or [FN-y antibody. Neutralization of RANTES， induced by 8. abortus infection， served to prevent abortion. These results indicate that the pro-duction and function of RANTES are correlated with TFN-y in pregnant mice infected with 8. abortus. KEY WORDS: abortion， 8rucella abortus， IFN-y， RANTES

Brucellosis is a serious debilitating disease in humans and

results in abortion and sterility in domestic animals. The eti-

ologic agents of brucellosis are Brucella spp.， small gram-negative and facultative intracellular pathogens that can

multiply within professional and non-professional phago

cytes [8， 9]. 1n contrast to other intracellular pathogens， Brucella species do not produce巴xotoxins，antiphagocytic

capsules or thick cell walls， resistant forms or fimbriae， and

do not show antigenic variation [14]. A key aspect of the

virulence of Brucella is its ability to proliferate within pro-

fessional and non-professional phagocytic host cells，

thereby successfully bypassing the bactericidal effects of

phagocytes， and their virulence and chronic infections are thought to be due to their ability to avoid the killing mecha-

nisms within host cells [20， 28]. Infection in humans is

almost exclusively due to zoonosis， either through direct

contact with infected animals or from contaminated dairy

products [19]. The mouse model， particularly that using the

non-pregnant mouse， has been used extensively to study some aspects of the pathogenesis of brucellosis [11]. While

brucellosis is known to primarily affect the reproductive

tract in the natural host and has been much studied， little is

known regarding the cellular and molecular mechanisms of

Brucella infection in the pregnant mouse [27]. The infec-

tious abortion model using the pregnant mouse is a powerful

tool for investigating the mechanisms of Brucella pathogen-

esis， and in our previous study we demonstrated that B. αbortus causes abortion in pregnant mice by inoculating

bacteria on day 4.5 of gestation [15]. We found that there

* CORRESPONDENCE TO: WATARAI， M.， Department of Veterinary Public Health， Faculty of AgricuItur巴，Yamaguchi University，

1677-1 Yoshida， Yamaguchi 735-8515， Japan e-mail:watarai@yamaguchi-u.ac.jp

1. Vet. Med. Sci. 70(7): 681-686， 2008

was a higher degree of bacterial colonization in the placenta

than in other organs， that there were many bacteria in tro-

phoblast giant (TG) cells in the plac巴ntaand that an intrac-

ellular replication-defective mutant did not induce abortion.

Transient interferon-y (1FN-y) production induced by infec-

tion with B. abortus also contributes to infectious abortion， and its neutralization served to prevent abortion.

It has been suggested that during normal pregnancy a

shift from a Th 1-to a Th2-polarized immune response

al10ws the survival ofthe fetus [21]. Th2 cytokines such as

TGF-s， 1L-4 and IL-I 0 ar巴 notonly able to prevent immu-

nopathologic events， but also hav巴beneficialeffects in suc-

cessful pregnancy [7]. It has also been suggested that

cytokines and chemokines play a role in the pathogenesis of

idiopathic recurrent spontaneous abortion [7]， but their role

in abortion induced by B. abortus infection is still unclear. Regulated upon activation normal T-cell expressed and

secreted (RANTES) is a chemokine that has recently been

implicated in trophoblast and spermatozoa migration in

view of its well-established chemoattractant properties [17，

26]. 1n the present study， we investigated the pathogenesis

of B. abortus-induced abortion in pregnant IFN-y knockout

mjce and noted that RANTES plays an important role in this

process.

MATERIALS AND METHODS

Bacterial strains: All B. abortus derivatives were from

544 (ATCC23448) smooth virulent B. abortus biovar 1

strains. Ba598 (544dvirB4) was also used in this study [30， 31]. B. abortus strains were maintained as frozen glycerol

stocks and cultured on Bruc巴lIabroth (Becton Dickinson，

NJ， U.S.A.) or Brucella broth containing 1.5% agar.

K. WATANABE ET AL

mouse RANTES monoclonal antibodies (clone 53405) or

IFN-y monoclonal antibodies (clone HB 170)， administering 200μg of the respective antibody in a volume of 0.1 ml

intraperitoneally 24 hr before infection. As a control， mice were inj巴ctedwith 200μg of normal rat IgG in 0.1 ml

according to the same schedule as for the mice treated with

anti-RANTES or antトIFN-ymonoclonal antibodies. Bacte

rial infection was achieved as described abov巴. Blood was

collected at 1，3，5 or 7 days after infection， and serum 1巴velsof RANTES and IFN-y were measured with an ELISA kit as

described above. On day 18.5 of gestation， a judgment was

made as to whether mice were pregnant or not as described

above

Statistical analysis: AII statistical analysis was conducted

using the Student' s t test

B. abortus infection in IFN-y knockout mice: We previ-

ously reported that transient induction of IFNγproduction

is a key event in abortion induced by B. abortus infection

[15]. In order to clarify the contribution of IFN-y to infec-

tious abortion， pregnant IFN-y knockout mice were infected with B. abortus， and abortion rates were examined. As

reported previously， pregnant normal mic巴 remam巴dalive

after B. abortus infection (Fig. IA)， but all B. abortus infected pregnant IFN-y knockout mice died within 15 days

of infection (Fig. 1 A). Bact巴rialgrowth was cxamined in

the spleens of infected pregnant IFN-y knockout mice to

determin巴ifbacterial colonization was predominantly in the

spleen. Colonization by B. abortus was much greater in the spleens of pregnant IFN-y knockout mice than in those of

pregnant normal mic巴 (Fig.1 B). B. abortus induced sple-

nomegaly as a consequence of the host inflammatory

response in the pregnant normal mice， but splenomegaly was not induced in the pregnant IFN-y knockout mice (Fig.

LC).

Transient increase in RANTES in pregnant 1 FN-y knock-

RESULTS

682

Mice: Six to ten-week-old BALB/c female mice were

individually mated to 6-to 10-w巴巴k-oldBALB/c male mice.

All of these mice were obtained from CLEA Japan (Tokyo，

Japan). The normal gestational time for these mice is 19

days， and the vaginal plug was observed on day 0.5 of ges-

tation. In addition， six to eight-week-old IFN-y knockout

mice on the BALB/c background were obtained from Jack-

son Laboratories (Bar Harbor， ME， U.S.A.). The animal

experiments were permitted by Animal Research Commit-

tee of Obihiro University of Agriculture and Veterinary

M巴dicine.

Virulence in pregnant mice: Groups of three or five preg-nant mice were infected intraperitoneal1y with approxト

mately 104 CFU of brucellae in 0.1 ml saline at the indicated

days of gestation [15]. On day 18.5 of gestation or when

mice died， their fetus， placenta， and spleen were removed and homogenized in saline. Tissue homogenates were seri-

ally diluted with PBS and plated on Brucella agar to count

the number of CFU in each organ. Fetuses were determined

to be alive if there was a heartbeat， and dead if there was no

heartbeat.

Cytokine measurement: S巴rumlev巴lsofRANTES， TFN-y

and MCP-I were measured for infected and uninfected vir-

gin and pregnant mice. To achieve infection， groups of five

mice were inoculated intraperitoneally with approximately

104 CFU of brucellae in 0.1 ml saline on day 4.5 of gesta-

tion， and blood was collected at 1， 3， 5 or 7 days after infec-

tion. Blood was collected at the same times for uninfected

mice. On day 18.5 of gestation， uteri were removed， and a

judgment was mad巴 asto whether mice were pregnant or

not. Serum levels of RANTES， IFN-y and MCP-I were

measured with an enzyme linked immunosorbent assay

(ELISA) kit (PIERCE Endogen， Rockford， IL， U.S.A.) according to the instructions of the manufacturer. The

cytokine antibody array used was obtain巴dfrom RayBio-

tech (Norcross， GA， U.S.A.).

ln vivo deplelion of RANTES and IFN-y: RANTES and

IFN-y were neutralized in the mice through the use of ant卜

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Fig. 1. B.aborlus infection in pregnant IFN-y knockout mice. Pregnant (lFN-y KJO pregnant) and non-pregnant (IFN-y KJO non-pregnant) TFN-yknockout mice and pregnant normal mice (BALB/c pregnant) were infected with B. aborlus. The fig-ure shows number of mice (A)， bacterial growth in spleen (B) and spleen weight (C)。

683 RANTES IN INFECTIOUS ABORTION

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Fig. 2. lnduction of RANTES in pregnant IFN-y knockout mic巴 byB. abortus infection. RANTES (A)， and MCP-I (B) serum lev巴Iswere measured in each mous巴byELlSA at the indicated numbers of days after infection. The means and SE for groups of 5 mice are shown. StatisticaIly significant differences betw巴enpregnant and non-pregnant mice are indicated by asterisks (*， P<O.OI).

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Fig. 3. Transient increase in RANTES in normal mice induced by B. aborTus infection. Pregnant and non-pregnant normal mice were infected with wild type (WT) or virB4 mutant (virB4). RANTES (A)，

and MCP-I (B) serum levels were measured in each mouse by ELlSA at the indicated numb巴rsof days after infection. The means and SE for groups of five mice are shown. Statistically significant differ-ences between mic巴inf，巴ctedwith and without wild type B. abortus are indicated by asterisks (*， P<O.OI).

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Days after infection

tion due to B. abortus infection， we next examined RANTES production in normal mice with B. abortus infec-tion in detaii. For wild type B. abortus inf，巴ction，RANTES was induced within 3 days of infection in pregnant mice， but the start of induction was 2 days later in non-pregnant mice.

There was no induction of RANTES in pregnant and non-pregnant mice infected with virB4 mutant， which do巴snot have the ability of intraceilular replication (Fig. 3A). No significant MCP-l production was observed in pregnant or non-pregnant mice infected with the wild type or virB4 mutant (Fig. 3B). These results suggest that the type IV secretion system contributes to RANTES production

Preventing abortion by neutralizing RANTES: To deter-mine if abortion is prevented by neutralizing the RANTES produced as a result of bacteriai infection， pregnant mic巴

out and normal mice induced by B. abortus infection: To examine cytokine patterns in B. abortus infected pregnant lFN-y knockout mice， we conduct巴drough screening using a cytokine antibody array. As this showed that RANTES was greatly induced， we decided to determine if RANTES contributes to induction of abortion by B. abortus infection by using ELISA to measure the production of RANTES in pregnant mice. As a resuit， we observed a large amount of induced RANTES production in pregnant IFN-y knockout mice at 3 days after infection with B. abortus， but afterwards RANTES production decreased rapidly (Fig. 2A). No sig-nificant MCP-l production was observed in infected preg-nant IFN-y knockout mice or pregnant normal mice (Fig. 2B).

As these results imply that RANTES contributes to abor-

684 K. WATANABE ET AL.

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Fig.4. Neutralization of RANTES and IFN-y using monoclonal antibodies. RANTES and IFN-y W巴ren巴utralizedin the mice by administering antトmouseRANTES or IFN-y monoclonal antibodies，

respectively， and then the mice wer巴 infectedwith wild type B. abortus (WT). RANTES (A) and IFN-y (B) were measured by ELlSA. The means and SE for groups of 5 mice are shown. Statist卜

cally significant differences between the untreated control and antibody treated mice are indicat巴dby asterisks (*， P<O.O 1)

were inoculated with monoclonal anti-RANTES antibodies

or anti-IFN司 yantibodiesI day before infection with B.αbor-tus. Thereafter， serum levels of RANTES and IFN-y， and

abortion rates were recorded. Induction of RANTES and

IFN-y production was observed within 3 days of infection in

pregnant mice， and this was inhibited by inoculation with

RANTES or IFN-y antibodi巴s，respectively (Fig. 4A and B)

Prior inoculation with anti-RANTES antibodies was also

observed to inhibit abortion as compared to non-inoculated

mice (Fig. 5A). However， there was no significant differ-

ence betwe巴npregnant mice inoculated with anti-RANTES

antibodies and non-inoculated pregnant mice as regards

bacterial growth in the spleen (Fig. 5B).

DISCUSSION

In the present study， we found that RANTES production

contributes to abortion due to B. abortus infection in preg-nant mice. We also noted that the production of RANTES

and IFN-y are influ巴nc巴dby each other in pregnant mice.

Our previous study showed that a transient increase in IFN-

ydu巴toBrucella infection causes abortion in pregnant mice [15]. For many other intracellular bacterial and protozoan

pathogens， it has been shown that IFN-y is an important

compon巴ntof Th 1 immune responses and contributes to

control through its ability to activate macrophages to

enhance microbial killing. The role of IFN-y in the control

of B. abortus infections has been demonstrat巴dby adminis-

tering recombinant IFN-y to BALB/c mice. This tr巴atment

resulted in a 10-fold d巴creasein the number of bacteria at 1

week after infection [24]. Similarly， the neutralization of

endogenous IFN-y through the administration of anti-lFN-y

monoclonal antibodies resulted in a decr巴asein control of B.

abortus infection [35]. IFN-y is important for control of B abortus infection in BALB/c mice during the first week of infection， since an increase in bacteria has been measured in

IFN-y knockout mice at I week post infection [12， I3].

However， by 3 weeks post infection， the number of bacteria

recovered was equivalent regardless of whether IFN-y was

present or not， and BALB/c mice survived for 10 weeks when IFN-ywas knocked out [18].

ln the present study， pregnant lFN-y knockout mice died

within 15 days of B. abortus infl巴ction.Pregnancy leads to a

generalized suppression of the adaptive immune system，

typified by significantly decreased cell-mediated immunity

and reduced Th 1 responsiveness [21， 33， 34]， and this

immunosuppressed state prevents matemal rejection of the

fetus but has the unfortunate consequence of increasing

matemal susceptibility to certain infectious agents [16， 23].

This is consider巴dto be the reason that pregnant IFN-y

knockout mice died mor巴rapidlydue to B. abortus infl巴cttOnthan non-pregnant IFN-y knockout mice

Immune cells and their secretory products have be巴nrec-

ognized as important pathophysiological mediators of recur-

rent spontaneous abortions and endometriosis in humans [7，

25，32]. On the other hand， chemokines are involved in T-

cell trafficking during normal processes and also in patho-

logical events such as inflammation and endothelium dam-

age [29]. RANTES is a chemokine that has recently been

implicated in trophoblast and spermatozoa migration in

view of its well-established chemoattractant properties [17，

26]. Th巴 specificability of RANTES to downregulate T-

cell responses suggests it might be relevant for fetal toler-

anc巴 induction，but other than this no role of RANTES in

pregnancy has been established [22]. A recent study

reported that the secretion ofRANTES， macrophage inflam-

RANTES lN INFECTIOUS ABORTION 685

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Fig. 5. Preventing abortion by neutralizing RANTES. RANTES was neutralized in the mice by administering anti-mouse RANTES mon-oclonal antibodies， and th巴nthey were infected with wild type (WT) or νirB4 mutant (νirB4). The figure shows number of fetuses (A)， and bacterial growth in the spl巴enand placenta (B)

matory protein (MIP)ーlα，MIP-I s， and activated-induced， T

cell-d巴rIved，and chemokine-related cytokin巴 (ATAC)in

polyclonally activat巴dNK and T cells was closely associ-

ated with the secretion of IFN-y [10]. RANTES is not only

co-secreted to a great extent with IFN-yat th巴 single-cell

level but also synergize functionally with IFN-y on a com-

mon target population [10]. In lhis s巴nse，th巴 production

and function of RANTES correlate with IFN-y in B. abortus

infect巴dpregnant mice. Further， RANTES may act as a

component of Th 1 immune responses with IFN-y in preg-

nant町lIce.

NK cells are large granular Iymphocytes found in periph-

eral blood and also in the maternal decidua during preg-

nancy. The actions of NK cells on trophoblast lineage cells

are likely mediated by NK cell secretory products， including cytokines， and may be direct or indirect. Uterine NK cells

produce several cytokines and are the primary sourc巴 of

IFN-y in the metrial grand [1，2]. IFN-y has been implicated

as a major mediator of uterine NK cell function during preg-

nancy [3， 4]. Trophoblast cells are among a variety of diι

ferent IFN-y targets， and in vitro trophoblast cell differentiation， survival， and outgrowth are affected by IFN-Y [1，5]. However， th巴 functionof IFN-y is more diverse

than the induction of bact巴ricidalfunction and includes the

stimulation of antigen presentation through class 1 and class

II MHC molecules， the orchestration of leukocyte-endothe-lium interactions， the effects on cell proliferation and apop-tosis， as well as stimulation and repression of a variety of

genes whose functional significance remains obscure [6].

NK cells may play a role in controlling brucellosis in preg-

nant mice and shift from Th2-to Thl-polarized immune

response may induce the abortion by B. abortus infection.

ACKNOWLEDGEMENTS. We thank Dr. Alexander Cox

for his critical reading of the manuscript and Dr. Yoshitaka

Omata for his valuable suggestions. This work was sup-

port巴d，in part， by grants from Program for Promotion of Basic Research Activities for Innovative Bioscience， and from Scientific Research (19041006 and 18790298)， Japan

Society for the Promotion of Scienc巴.

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