expression of natural cytotoxicity receptors and a2v-atpase on peripheral blood nk cell subsets in...

Expression of Natural Cytotoxicity Receptors and a2V-ATPaseon Peripheral Blood NK Cell Subsets in Women with RecurrentSpontaneous Abortions and Implantation FailuresAtsushi Fukui1,2, Evangelos Ntrivalas1, Alice Gilman-Sachs1, Joanne Kwak-Kim1,3, Sung-Ki Lee1,3,4, RitaLevine1, Kenneth Beaman1

1Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science/The Chicago Medical School, North Chicago, IL,

USA;2Department of Obstetrics and Gynecology, Hirosaki University School of Medicine, Hirosaki, Aomori, Japan;3Department of Obstetrics and Gynecology, Rosalind Franklin University of Medicine and Science/The Chicago Medical School, North Chicago, IL,

USA;4Department of Obstetrics and Gynecology, Konyang University College of Medicine, Daejeon, Korea

Introduction

Natural killer (NK) cells are likely to play an import-

ant role in human pregnancy.1,2 NK cells compose

5–10% of peripheral blood lymphocytes (PBLs) and

are distinguished from other cell types by the

expression of NK specific surface markers, i.e. CD56

positive cells. Human NK cell subsets exist as either

as CD56bright or CD56dim NK cells. In addition, a

number of natural killer T (NKT) cells are found in

Keywords

a2V-ATPase, CD56, NKp46, NKp44, NKp30,

NKT

Correspondence

Atsushi Fukui, Department of Microbiology and

Immunology, Rosalind Franklin University, 3333

Green Bay Rd, North Chicago, IL 60064, USA.

E-mail: [email protected]

Submitted July 21, 2006;

accepted August 16, 2006.

Citation

Fukui A, Ntrivalas E, Gilman-Sachs A, Kwak-Kim

J, Lee S-K, Levine R, Beaman K. Expression of

natural cytotoxicity receptors and a2V-ATPase

on peripheral blood NK cell subsets in women

with recurrent spontaneous abortions and

implantation failures. Am J Reprod Immunol

2006; 56:312–320

doi:10.1111/j.1600-0897.2006.00431.x

Problem

Natural cytotoxicity receptors (NCRs) are unique markers, which regu-

late NK cell cytotoxicity and cytokine production. a2V-ATPase is

expressed on subsets of PBMC and regulates the extracellular environ-

ment, which facilitates NK cytotoxicity or cytokine secretion. In this

study, we aim to investigate the expression of NCRs and a2V-ATPase in

peripheral blood NK cells of women with recurrent spontaneous abor-

tions (RSA) or implantation failures.

Method of study

Peripheral blood NK cells (CD56dim and CD56bright were analyzed for

the expression of NCRs (NKp46, NKp44 and NKp30) and a2V-ATPase

using 3-color flow cytometry in women with RSA (n¼24), implantation

failures (n¼19) or normal healthy women (n¼13).

Results

CD56+/NKp46+ cells were markedly decreased (P<0.05) and CD56bright/

a2V-ATPase+ cells were significantly increased (P<0.05) in women with

RSA as compared to those of normal controls. In women with RSA or

implantation failures, expression of NKp46, NKp44, NKp30, and

a2V-ATPase on CD56bright NK cells was significantly up-regulated as

compared with those of CD56dim NK cells.

Conclusion

The differential expression of NCRs and a2V-ATPase in NK cell subsets

may suggest dysregulation of NK cytotoxicity and cytokine production

in women with RSA and implantation failures.

ORIGINAL ARTICLE

American Journal of Reproductive Immunology 56 (2006) 312–320 ª 2006 The Authors

312 Journal compilation ª 2006 Blackwell Munksgaard

peripheral blood and CD56bright cells are mainly in

the decidua or placenta. In peripheral blood, the

main population of NK cells is CD56dim cell. Studies

suggest that systemic regulation of peripheral blood

leukocytes contribute to reproductive success.3,4

Natural cytotoxicity receptors (NCRs), which

include NKp30, NKp44 and NKp46, are expressed

exclusively on NK cells. The NKp46 receptor is a 46-

kDa type I membrane glycoprotein characterized by

two C2-type Ig-like domains in the extracellular por-

tion. The NKp44 receptor is a 44-kDa type I mem-

brane glycoprotein characterized by a single V-type

Ig-like domain in the extracellular portion. The

NKp30 receptor is a 30-kDa type I membrane glyco-

protein characterized by a single V-type Ig-like

domain in the extracellular portion. NCRs are the

major receptors involved in NK cytotoxicity. NKp46

and NKp44, but not NKp30, recognize viral proteins

such as the haemagglutinin of influenza, or the

haemagglutinin-neuraminidase of parainfluenza

virus.5,6 The endogenous cellular ligands recognized

NCRs are not known. The NKp30 and NKp46 recep-

tors are expressed on the surface of activated and

non-activated NK cells. However, the NKp44 recep-

tor is expressed on the surface of activated NK cells

only. In addition, the NKp30 and NKp46 receptors

have a function in cytotoxic activity and cytokine

production of NK cells.

The a2-isoform of proton-pumping vacuolar AT-

Pase (a2V-ATPase), formerly termed TJ6 or regener-

ation and tolerance factor (RTF) is a relatively newly

characterized marker of activation. It is a 70-kDa

protein that spans the membrane seven times and is

abundantly expressed in the developing thymus.7,8

a2V-ATPase is expressed in the cell membrane and

can regulate the pH of the extracellular environ-

ment,9,10 which may facilitate NK cell killing or cy-

tokine secretion. a2V-ATPase is expressed on B cells

during normal pregnancy, and on NK cells during

abnormal pregnancy in peripheral blood.11–13

It is suggested that implantation-related immune

abnormality exists in some forms of infertility and in

women who experienced recurrent spontaneous

abortions (RSAs).1,2,14–17 We previously reported

that the increase of cytotoxic NK cells in peripheral

blood and the endometrium may have an effect on

the therapeutic results of in vitro fertilization and

embryo transfer (IVF-ET).14 This may be related to

the imbalance of killer immunoglobulin-like recep-

tors in peripheral blood NK cells and may affect the

reproductive outcome.18 This activity in reproduc-

tion is only beginning to be studied. To begin to

reveal the participation of NCRs and a2V-ATPase, we

undertook a flow cytometric analysis of NCRs and

a2V-ATPase on peripheral blood NK cells of women

with RSA or implantation (IVF-ET) failures.

Materials and methods

Study Subjects

The study design was a prospective study. Study sub-

jects were recruited at Reproductive Medicine Pro-

gram from October 1, 2005 to June 30, 2006. All

study subjects had signed informed consents prior to

entering the study, in accordance to local IRB proto-

col.

We classified patients by their histories as fol-

lows: the RSA group (n ¼ 24), women with a his-

tory of three or more spontaneous abortions; the

implantation failures group (n ¼ 19), women with

a history of two or more IVF-ET failures in spite of

good embryo transfer; controls are women with

normal fertile history (n ¼ 13). Women with ana-

tomical, infectious or chromosomal causes of RSA

were excluded from this study. Women with evi-

dent endometriosis, anatomical or infectious etiolo-

gies of implantation failures were also excluded.

Blood was drawn from study groups prior to any

treatment.

Flow Cytometric Analysis of the Peripheral Blood

Lymphocytes

The following monoclonal antibodies (mAbs) were

used to analyze the surface antigens of peripheral

blood leukocytes: anti-CD3 ECD (Beckman Coulter,

Miami, FL, USA), anti-CD56 FITC (BD Biosciences,

San Jose, CA, USA), anti-CD335 (NKp46) PE (BD

Biosciences), anti-CD336 (NKp44) PE (Beckman

Coulter), anti-CD337 (NKp30) PE (Beckman Coul-

ter), and anti-a2V-ATPase (2C1) PE.19 Labeling of

peripheral blood leukocytes with this panel of

mAbs was performed using standard techniques.

Briefly, 100 lL of whole blood was incubated with

mAbs for 20 min at room temperature, and cells

were lysed and fixed, and the cells were washed

twice in phosphate-buffered saline. Immunofluores-

cence and three-color flow cytometric analysis

were performed using a FACScalibur flow cytome-

ter (Becton Dickinson, San Jose, CA, USA) with

computer interfacing to BD cell QuestPro for

NCR AND A2V-ATPASE ON NK CELLS IN RSA WOMEN


Journal compilation ª 2006 Blackwell Munksgaard 313

full-list-mode data storage, recovery, and analysis.

A gate was set on the lymphocyte region by

characteristic forward and side scatter parameters.

For each sample, 104 PBLs were evaluated. The

percentage of CD56+ cells or subsets of CD56+ cells

expressing each NCR or a2V-ATPase was deter-

mined by flow cytometry. The percentage of NCR+

cells or a2V-ATPase+ cells/CD56+ or subsets of

CD56+ cells was calculated from this data. We

distinguished CD56dim and CD56bright NK cells

according to their fluorescence, and defined NKT

cells as CD3+/CD56+ cells.

Statistical Analysis

All data are presented as the mean ± S.D. The data

were analyzed using StatView version 4.5 (Abacus

Concepts, Inc., Berkeley, CA, USA). Age distribution

of study groups and controls were analyzed by one-

way anova. Expression of NCRs and a2V-ATPase in

peripheral blood NK cell subsets were analyzed by

Fisher’s exact probability test of frequency distribu-

tion. Different expression of NCRs and a2V-ATPase

between CD56dim and CD56bright NK cells were ana-

lyzed by paired t-test. Differences were considered

significant for probability <0.05.

Results

Study Population

Patient characteristics including age, number of RSA

and implantation failures are shown in Table I.

There are no differences in age distribution among

women with RSA, implantation failures and normal

controls.

Expression of NCRs and a2V-ATPase in Peripheral

Blood NK Cell Subsets

Representative dot plots of the expression of NCRs

(NKp46, NKp44, NKp30) or a2V-ATPase on CD56+

NK cells and NK cell subsets for RSA patient and

normal healthy fertile controls are shown in Fig. 1.

Flow cytometric analysis of NCRs and a2V-AT-

Pase expression in peripheral blood NK cells of the

RSA group, the implantation failures group and

controls are shown in Fig. 2. For the expression of

NKp46, the percentage of CD56+/NKp46+ cells in

the RSA group (53.5 ± 27.4%) was significantly

lower than that in controls (71.4 ± 13.8%)

(P < 0.05) (Figs 2a and 3). Fig. 3 is a representative

dot plots which demonstrate the difference of

NKp46 expression on NK cells in RSA patient and

normal healthy fertile control. There were no signi-

ficant differences among the three groups for the

expression of NKp46 on CD56dim, CD56bright NK

cells and NKT cells. For the expression of NKp44

and NKp30 on NK cells (CD56+, CD56dim,

CD56bright) and NKT cells, there were no significant

differences among three groups (Fig. 2b,c). For the

expression of a2V-ATPase, the percentage of

CD56bright/a2V-ATPase+ cells in the RSA group

(18.4 ± 20.1%) was significantly higher than that

in controls (6.4 ± 9.3%) (P < 0.05) (Figs 2d and 4).

Fig. 4 shows the difference of the expression of

a2V-ATPase on NK cells between RSA patient and

normal healthy fertile control. There were no signi-

ficant differences among the three groups for the

expression of a2V-ATPase on CD56+ and CD56dim

cells and NKT cells.

Different Expression of NCRs and a2V-ATPase

between CD56dim and CD56bright NK Cells

For women with RSA, there were significant differ-

ences in the expression of NKp46 between CD56dim

(46.5 ± 32.7%) and CD56bright (84.1 ± 24.6%)

(P < 0.001), NKp44 between CD56dim (0.3 ± 0.5%)

and CD56bright (9.8 ± 13.8%) (P < 0.05), NKp30

between CD56dim (36.6 ± 30.7%) and CD56bright

(62.3 ± 31.4%) (P < 0.01), and a2V-ATPase


(16.6 ± 15.1%) (P < 0.01) NK cells, respectively

(Fig. 5a).

Table I Age, Obstetrical and Infertility Histories of Women with

Recurrent Pregnancy Losses, Implantation Failure and Normal

Controls

RSA (n ¼ 24)

(mean ± S.D.)

Implantation

failures

(n ¼ 19)

(mean ± S.D.)

Controls

(n ¼ 13)

(mean ± S.D.)

Age (years) 35.3 ± 4.8 33.6 ± 3.6 37.7 ± 9.8

No. of pregnancies 4.0 ± 1.9 0.3 ± 0.6 1.7 ± 0.9

No. of deliveries 0.6 ± 0.8 0.1 ± 0.2 1.7 ± 0.9

No. of spontaneous

abortions

2.8 ± 1.4 0.1 ± 0.3 0.0 ± 0.0

RSA, recurrent spontaneous abortion.

FUKUI ET AL.



For women with implantation failures, there were

significant differences in the expression of NKp46


(84.8 ± 22.1%) (P < 0.0001), NKp44 between

CD56dim (1.0 ± 1.6%) and CD56bright (14.8 ± 22.2%)

(P < 0.02), NKp30 between CD56dim (39.7 ± 26.2%)

and CD56bright (55.7 ± 30.6%) (P < 0.01), and a2V-

ATPase between CD56dim (2.8 ± 3.5%) and

CD56bright (22.9 ± 15.1%) (P < 0.01) NK cells,

respectively (Fig. 5b).

For normal healthy controls, there were signi-

ficant differences in the expression of NKp46

Fig. 1 Representative dot plots of the expres-

sion of natural cytotoxicity receptors on Nat-

ural Killer cells in a woman with RSA and a

normal healthy woman. A gate is set on the

lymphocytes. Lymphocytes are classified into

CD56+ and CD56) cells. CD56+ cells are

classified into CD56bright and CD56dim cells.

Co-expression of CD56 with NCRsor

a2V-ATPase is evaluated.





(92.5 ± 16.3%) (P < 0.01), and of NKp44 between

CD56dim (0.9 ± 0.7%) and CD56bright (15.1 ± 14.2%)

(P < 0.05). However, there was no difference in

the expression of NKp30 between CD56dim (42.5 ±

30.2%) and CD56bright (49.6 ± 25.6%), or a2V-ATPase

Fig. 2 Flow cytometric analysis of natural cy-

totoxicity receptors and a2-isoform of proton-

pumping vacuolar ATPase expression on per-

ipheral blood natural killer cells in women

with recurrent spontaneous abortion, implan-

tation failures and controls.

The ordinate indicates the percentage of NCR

or a2V-ATPase positive cells in each subset of

NK cells.

(a) Expression of NKp46 in NK cells in women

with RSA, implantation failures and controls.

(b) Expression of NKp44 in NK cells in women


(c) Expression of NKp30 in NK cells in women


(d) Expression of a2V-ATPase in NK cells in

women with RSA, implantation failures and

controls.

FUKUI ET AL.



between CD56dim (1.8 ± 1.5%) and CD56bright (7.9 ±

11.5%) NK cells, respectively (Fig. 5c).

Discussion

We have previously shown that peripheral blood NK

cell cytotoxicity was elevated in women with RSA20

and peripheral blood NK cells of women with RSA

had increased expression of CD69 and decreased

expression of CD94.15 At the time of embryo trans-

fer, elevated NK cell cytotoxicity correlates with the

resultant abortion following IVF-ET cycle.14 These

results suggested that activated peripheral blood NK

cells might cause an unfavorable pregnancy result.

Often the interaction between peripheral blood

NK cells and trophoblasts are not well understood,

and researchers are investigating with various roles

of peripheral and decidual NK cells.21,22 NK cells

preferentially kill targets with lower expression of

major histocompatibility complex (MHC) class I pro-

teins, because fewer inhibitory receptors are enga-

ging ligands. As a consequence, syncytiotrophoblasts

are not free from peripheral blood NK cell cytotoxici-

ty. However, if trophoblast cells produce cytokines

that lowered MHC class I levels on NK cells, the pre-

dicted consequence would be enhanced immunosup-

pression. The absence of allo-MHC on the NK

effectors could result in a larger pool of trans-reactive

inhibitory receptors and a consequent inhibition of

CD56dim CD56bright

CD56dim CD56bright

CD56

64pK

N

Normal healthy woman

Women with RSA

17.3%

48.8%

51.2%

82.7%

Fig. 3 Representative dot plots of the expression of NKp46 on natural

killer cells in a woman with recurrent spontaneous abortion and a

normal healthy woman. Numbers in solid line show the percentage of

CD56+NKp46+ or CD56+NKp46) cells in total CD56+ cells.

CD56dim CD56bright

CD56dim CD56bright

CD56

100 101 102 103

CD56 FITC10410

010

110

210

3

RT

F(2C

1) P

ER

TF(

2C1)

PE

104

100

101

102

103

104

100 101 102 103

CD56 FITC104

a2V

-AT

Pase

Normal healthy woman

Women with RSA

98.5%

22.2%

77.8%88.3%

11.7%

1.2%

98.8%

1.5%

Fig. 4 Representative dot plots of the expression of a2-isoform of

proton-pumping vacuolar ATPase on natural killer cells in a woman

with recurrent spontaneous abortion and a normal healthy woman.

Numbers in solid line show the percentage of CD56dima2VATPase+ or

CD56dima2VATPase) cells in total CD56dim cells. Numbers in dotted

line show the percentage of CD56brighta2VATPase+ or CD56brighta2VAT-

Pase) cells in total CD56dim cells.




alloreactivity.23 Studies of NK cell receptor expres-

sion highlight the in vivo regulation of NK cytotoxici-

ty during implantation and pregnancy.

Natural killer cells express a number of inhibitory

receptors that recognize the MHC class I molecules

expressed on normal cells.24 The lack of expression

of one or more MHC class I alleles or the expression

of insufficient amounts of class I molecules leads to

NK-mediated target-cell lysis.25 Trophoblast cells

temporally express the HLA-C, HLA-G and HLA-E

MHC class I molecules, which are candidate ligands

for decidual NK cells. HLA-C binds to KIR2DL1 and

KIR2DL2 on NK cells. We previously reported that

women with implantation failures have decreased

expression of killer inhibitory receptors (CD158a and

CD158b) compared with normal healthy controls.18

We believe that the balance between inhibitory and

activating receptor expression on NK cells may be

key to a successful pregnancy.

In this study, we provide the experimental evi-

dence that the surface expression and functional

change of NCRs in peripheral blood have a relation

to the reproductive success. NKp46, NKp44 and

NKp30 are the first three members of NCR family

and these receptors are found only on NK cells.26–28

NKp46 is expressed at higher levels on CD56 cells

than NKp44, NKp30 or a2V-ATPase. Both peripheral

blood NK cells and decidual NK cells express NCRs.29

However, the function of these receptors during

pregnancy is not known.

In this study, there was a significant difference in

the expression of NKp46 on NK cells between the

RSA group and controls. The expression of NKp46

on CD56+ cells in the RSA group was significantly

lower than that of controls. NKp46 may function in

both cytotoxicity and cytokine production.30 Our

data suggest that cytokine production by NK cells in

the RSA groups is different than in controls. The cy-

tokine repertoire of peripheral NK cells is mainly

type 1 cytokines such as interferon-c and tumor nec-

rosis factor-a, but NK cells are capable of producing

type 2 cytokines such as interleukin (IL)-4, IL-5 and

IL-13.31,32 It is reported that there is another type of

NK cells, which produce TGF-b33 or IL-10.34 NK cells

which produce TGF-b are called NK3 cells and IL-10

producing cells are called NKr1 cells. These NK3 and

NKr1 cells are decreased in spontaneous abortion

cases.35 Our results demonstrate abnormalities of

NKp46 expression on CD56+ NK cells, which may

explain NK cell abnormalities of cytokine production

and cytotoxicity in women with RSA.

In this study, the percentage of CD56bright/a2V-AT-

Pase+ cells was significantly higher in the RSA group

when compared with controls, but there were no

significant differences in the percentage of CD56+/

a2V-ATPase+ cells and CD56dim/a2V-ATPase+ cells.

The presence of a2V-ATPase on maternal circulating

B cells and NK cells was previously shown to be

associated with the pregnancy outcome. Specifically,

normal karyotype nonviable pregnancies were char-

Fig. 5 Expression of natural cytotoxicity receptors and a2V-ATPase in

peripheral blood natural killer cell subsets of women with recurrent

spontaneous abortions, implantation failures and controls.

The ordinate indicates the percentage of NCR or a2V-ATPase positive

cells in CD56dim or CD56bright cells.

(a) Expression of NCRs in peripheral blood NK cell subsets in women

with RSA.

(b) Expression of NCRs in peripheral blood NK cell subsets in women

with implantation failures.

(c) Expression of NCRs in peripheral blood NK cell subsets in controls.

FUKUI ET AL.



acterized by a decreased expression of a2V-ATPase

on B cells and an increased expression of that on NK

cells.36 This study suggests that the NK cells, partic-

ularly CD56bright cells, are associated with the a2V-

ATPase associated pregnancy loss.

There were significant differences in the individual

expression of NCRs and a2V-ATPase between

CD56dim and CD56bright cells in both the RSA and

implantation failure group. However, there was no

difference in either NKp30 or a2V-ATPase in normal

controls. These data indicate that the increase of

NKp30 and a2V-ATPase expression on CD56bright

cells in the RSA group or the implantation failures

group and not in normal controls may mark a

dysregulation of cytotoxicity or cytokine production

in CD56bright NK cells.

Recently, it was reported that TGF-b1, which might

be released by tumors, neuroblastoma, carcinoma,

and leukemia, and is able to induce a strong down-

regulation of surface expression of triggering NK

receptors such as NKp30.37 It is also speculated that

the decrease of NKp30 on CD56dim cells in the RSA

group or implantation failures group may be due to

down regulation by TGF-b1.

In conclusion, NK cells have a major role in

human pregnancy. The differential expression of

NCRs and a2V-ATPase in NK cell subsets of

women with RSA and implantation failures may

be related to NK cell cytotoxicity and cytokine

production. We believe that successful modifica-

tions of NCRs, that is to say appropriate upregula-

tion of NKp46 on NK cells, could contribute to the

improvement of pregnancy outcome via proper

regulation of cytokine production. Further investi-

gation of NCRs and a2V-ATPase expression in NK

cell subsets in normal and abnormal pregnancies

will contribute to the better understanding of the

biology of NK cell in women with RSA and

implantation failures.

Acknowledgment

This study was partly supported by the Fund for the

Promotion of International Scientific Research,

Aomori Prefecture and City of Hirosaki, Japan.

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FUKUI ET AL.



expression of natural cytotoxicity receptors and a2v-atpase on peripheral blood nk cell subsets in...

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