original article: enhanced maternal anti-fetal immunity contributes to the severity of hypertensive...

Enhanced Maternal Anti-Fetal Immunity Contributes to theSeverity of Hypertensive Disorder Complicating PregnancyLi-Ping Liu1,2, Wei Huang3,4,5, Yue-Chao Lu1, Ai-Hua Liao1

1Family Planning Research Institute, Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology,

Wuhan, China;2Wuhan Women & Children Medical Healthcare Center, Wuhan, China;3Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China;4Key Laboratory of Organ Transplantation, Ministry of Education, Wuhan, China;5Key Laboratory of Organ Transplantation, Ministry of Health, Wuhan, China

Introduction

Hypertensive disorder complicating pregnancy

(HDCP), including pre-eclampsia and pregnancy-

induced hypertension or gestational hypertension, is

associated with significant morbidity and mortality for

both mothers and neonates.1 It adversely affects 2–

10% of pregnancies worldwide and remains a leading

cause of pregnancy-associated maternal and fetal

mortality and morbidity in developing countries.2,3

Keywords

Antibody-secreting cells, gestational

hypertension, monocyte activation,

pre-eclampsia

Correspondence

Ai-Hua Liao, No. 13, Hangkong Rd., 430030

Wuhan, China.

E-mail: [email protected]

Submitted October 20, 2009;

accepted December 4, 2009.

Citation

Liu L-P, Huang W, Lu Y-C, Liao A-H. Enhanced

maternal anti-fetal immunity contributes to

the severity of hypertensive disorder

complicating pregnancy. Am J Reprod

Immunol 2010; 63: 379–386

doi:10.1111/j.1600-0897.2009.00802.x

Problem

The aim of this study was to evaluate how fetal monocyte activation

and maternal anti-fetal antigen-specific antibody-secreting cells (ASC)

affect the severity of hypertensive disorder complicating pregnancy

(HDCP).

Method of study

Forty-six healthy third-trimester pregnant women and 20 patients with

gestational hypertension, 20 with mild pre-ecalmpsia and another

20 with severe pre-eclampsia were included in the study. Interleukin-6

(IL-6) release from cord blood monocytes was examined by intracellular

cytokine staining and flow cytometric analysis. Moreover, the maternal

anti-fetal antigen-specific ASC were detected by enzyme-linked immu-

nospot assay.

Results

A significantly increased percentage of IL-6-positive monocytes were

detected in the cord blood of study groups compared with the controls

(P < 0.01). The percentage of IL-6-positive monocytes was increased as

the disease progressed (P < 0.05). There were more anti-fetal antigen-

specific ASC in the study groups than those in the controls (P < 0.001).

Furthermore, the anti-fetal antigen-specific ASC showed difference in

gestational hypertensive and severe pre-eclamptic groups (P < 0.05).

Conclusion

We conclude that the fetal monocyte activation and the increase in

maternal anti-fetal antigen-specific ASC were related to the incidence

and severity of HDCP. These results provide both indirect and direct evi-

dence for the occurrence of exaggerated maternal humoral immunity

against the fetal antigens in HDCP.

ORIGINAL ARTICLE

American Journal of Reproductive Immunology 63 (2010) 379–386

ª 2010 John Wiley & Sons A/S 379

The pathophysiology of the disease involves not only

impaired trophoblast invasion, abnormal genetic

polymorphism, vascular endothelial cell activation,

immune intolerance by the maternal immune system

but also an exaggeration of a systemic inflammatory

process.4 However, the origin of HDCP, a disease

unique to pregnancy, is still matter of debate and has

not been fully elucidated.

Considerable evidence indicates that an immune

disorder accounts for the etiology of this disease. A

diverse number of immunologic abnormalities, such

as a decreased mixed-lymphocyte reaction between

the biologic parents of the fetus, T helper (Th) 1 pre-

dominance over Th2, dysregulation of multiple cyto-

kines, aberrant natural killer cell activation and

inadequate tolerance induction of regulatory T cells,

have been reported in HDCP.5–9

Previous findings showed that some pregnancy-

related diseases, such as pre-eclampsia or hemolysis,

elevated liver enzymes, low platelets (HELLP) syn-

drome, placental abruption and gestational diabetes,

are associated with pre-term activation of fetal

monocytes,10–12 indicating the existence of an

increased humoral immune response of the mother

against the semi-allogeneic fetus. Recently, our stud-

ies observed that both circulating memory B cells

and their capacity to generate into antibody-produc-

ing cells were increased in pre-eclampsia.13

In this study, we revisited interleukin-6 (IL-6)-

positive monocytes in cord blood as target cells to

evaluate whether the activation of fetal monocytes is

related to the proceeding of HDCP. Meanwhile, we

targeted the anti-fetal antigen-specific antibody-

secreting cells (ASC) to explore the potential cause

of the excessive maternal humoral immunity in

HDCP. The study aimed to provide more evidence

for the involvement of humoral immunologic fac-

tors, especially maternal anti-fetal immunity in

HDCP.

Materials and methods

Subjects

Pregnant women attending the Department of

Obstetrics and Gynecology at the Shangzhi Hospi-

tal, Wuhan, China, were recruited with informed

consent for the study approved by the local ethics

committees. The study group comprised 20

patients with gestational hypertension, 20 with

mild pre-ecalmpsia and another 20 with severe

pre-eclampsia at 36–40 weeks of gestation. Forty-

six healthy women with uncomplicated pregnancy

constituted the control group. These women in the

third trimester of normal pregnancy were matched

with the study group of similar age, gestational

age and parity (Table I). The definitions of gesta-

tional hypertension and mild or severe pre-eclamp-

sia have been described elsewhere.14 All

pregnancies in the study and control groups were

singleton.

Blood Sampling

Both maternal and cord blood samples from the nor-

mally pregnant women and the patients with HDCP

were collected into Vacutainer tubes (Becton Dickin-

son, Franklin Lakes, NJ, USA) containing ethylenedi-

Table I Clinical Characteristics and Percentage of IL-6-Positive Monocytes of Patients with Hypertensive Disorder Complicating Pregnancy and

Healthy Women in Third Trimester of Uncomplicated Pregnancy

Patient group

Normal term

pregnancy

(n = 46)

Gestational

hypertension

(n = 20)

Mild

pre-eclampsia

(n = 20)

Severe

pre-eclampsia

(n = 20) P-value

Age (years) 27.10 ± 4.57 28.00 ± 3.37 27.60 ± 3.42 28.00 ± 2.58 NS

Gestational age (weeks) 38.10 ± 1.13 37.80 ± 0.69 37.80 ± 0.92 37.90 ± 0.99 NS

Gravidity times 2.04 ± 1.11 3.17 ± 1.46 2.56 ± 1.06 2.88 ± 1.66 NS

Parity primipara (%) 71.74 67.14 68.89 66.67 NS

IL-6-positive monocytes (%)a 0.78 ± 0.54 4.38 ± 0.91 5.73 ± 2.18b 6.29 ± 2.71b <0.01

IL-6, interleukin-6.aThe estimated percentages of IL-6-positive monocytes are based on the CD14-positive monocyte population.bSignificantly increased amounts of IL-6-positive monocytes were detected for the mild pre-eclampsia group (P < 0.05) and for the severe pre-

eclampsia group (P < 0.01) in comparison with the gestational hypertensive group.

LIU ET AL.


380 ª 2010 John Wiley & Sons A/S

aminetetraacetic acid (EDTA). All the blood samples

were processed within 1 hr from being drawn.

Isolation of Cord Blood Mononuclear Cells

(CBMC)

Human venous umbilical cord blood (10 mL) was

collected into tubes containing EDTA from neonates

immediately after delivery. The EDTA blood was

diluted with 10 mL of phosphate-buffered saline

(PBS) and was gently layered on 10 mL Lympho-

lyte�-H (Cedarlane, Hornby, ON, Canada) with a

density of 1.077 g ⁄ mL. After centrifugation at

800 · g for 20 min, the CBMC were collected at the

interface and washed twice in 15 mL PBS. The cells

were immediately analyzed for the determination of

IL-6-producing monocytes using two-color flow

cytometry.

Flow Cytometric Analysis

The procedure of determination of IL-6-producing

monocytes in the cord blood followed the manufac-

turer’s instructions. Briefly, the 1 · 106 isolated

CBMC were directly stained with 0.5 mg of fluores-

cein isothiocyanate-conjugated monoclonal antibody

(mAb) specific for CD14 (Clone M5E2, IgG2a; BD

Bioscience, San Jose, CA, USA) expressed on mono-

cytes. Cells were washed twice with PBS supple-

mented with 0.5% fetal calf serum (FCS) and 5 mm

EDTA and centrifuged. Intracellular staining for IL-6

was performed using cell permeabilization reagents

purchased from Invitrogen Corporation (Carlsbad,

CA, USA). Briefly, the cells were fixed in 100 lL of

reagent A (fixation medium) and incubated for

15 min at room temperature. The cells were washed

once in 3 mL PBS + 0.1% NaN3 + 5% fetal bovine

serum. After the cell suspensions were centrifuged

for 5 min at 300 · g, the supernatants were aspi-

rated and the cell pellet was vortexed and fully

resuspended. Then the cells were permeabilized in

100 lL of reagent B (permeabilization medium) and

incubated for 20 min with 0.5 mg phycoerythrin-

conjugated rat anti-human IL-6 mAb (BD Biosci-

ence). Flow cytometric analysis was performed using

FACScalibur (BD Biosciences), and data were pro-

cessed using the CellQuest program (Becton Dickin-

son). Isotype-matched mouse IgG was used as

negative control.

Mononuclear cells were gated based on both for-

ward and side scatter characteristics (FSC and SSC).

The percentage of IL-6-positive monocytes was esti-

mated within the three different patient groups com-

pared with the control.

Isolation of Maternal Peripheral Blood

Mononuclear Cells (PBMC) and Pre-Stimulation

In Vitro

The isolation of the maternal PBMC was the same as

that of CBMC. After preparation, the PBMC were

cultured and pre-stimulated in vitro with pokeweed

mitogen (PWM, 5 lg ⁄ mL; Sigma, St Louis, MO,

USA) for 5 days. The in vitro culture conditions were

described previously.15 Briefly, the isolated maternal

PBMC were cultured at 37�C in a moist atmosphere

containing 5% CO2 at 7 · 105 cells ⁄ well (in 24-well

plates) in complete medium: RPMI1640 + 16% heat-

inactivated FCS supplemented with 2 mm l-gluta-

mine, 25 mm Hepes, 100 U ⁄ mL penicillin and

100 lg ⁄ mL streptomycin, 0.1 mm dispensable amino

acid solution (Sigma-Aldrich), 1 mm sodium pyru-

vate (Sigma-Aldrich) and 40 lg ⁄ mL apo-transferrin

(Sigma-Aldrich St.Louis, USA).

Preparation of CBMC Lysates

The CBMC lysates were prepared by incubating

3 · 106 PBS-washed viable cells in 50 lL lysis buffer

solution (150 mm Nacl, 20 mm Tris pH 7.2, 1% (v ⁄ v)

Triton X 100) for 10 min on ice. Then, the superna-

tants were collected following a brief micro-centri-

fuge spin (10,000 · g). The protein concentration in

the supernatants was determined by Bradford pro-

tein assay (Bio-Rad, Hercules, CA, USA) according to

manufacturer’s instructions. The CBMC lysates were

used as the origin of fetal antigens.

Enzyme-Linked Immunosorbent Spot-Forming

Cell (ELISPOT) Assay to Detect Anti-Fetal-Specific

Antibodies and Total Immunoglobulin-Secreting

Cells in Maternal Circulation

For the detection of maternal anti-fetal-specific anti-

bodies, the ELISPOT assay was used with a slight

modification as described previously.16 Briefly,

96-well multiscreen-HA plates (MAHAS4510; Milli-

pore, Bedford, MA, USA) were coated overnight with

70 lL ⁄ well of the CBMC lysate (0.25 mg ⁄ mL) at 4�C.

The plates were washed three times with PBS and

blocked with 150 lL of RPMI medium containing

3% bovine serum albumin (BSA) (Calbiochem, La

MATERNAL ANTI-FETAL IMMUNITY AND HDCP



Jolla, CA, USA) for 2 hr at 37�C in a moist atmo-

sphere containing 5% CO2. Pre-stimulated maternal

PBMC were washed and added to the plates in

serial double dilutions from 1 · 107 through

1.25 · 106 cells ⁄ ml (100 lL ⁄ well) and incubated for

24 hr at 37�C. Then the cells were discarded, and the

plates were washed six times with PBS containing

0.05% Tween20 (PBS-T). Negative controls are

obtained as follows. (1) The maternal pre-stimulated

PBMC were added to wells without the CBMC lysate.

(2) The plates were incubated with medium without

the maternal pre-stimulated PBMC. (3) Another

CBMC lysate from allogeneic fetus was coated. (4)

The plates were incubated with maternal pre-stimu-

lated PBMC from another allogeneic woman. To

detect bound secreted immunoglobulin molecules,

alkaline phosphatase-conjugated rabbit anti-human

IgG + IgM + IgA (H + L) (Jackson ImmunoResearch

Laboratories, PA, USA) diluted 1:1000 in PBS 0.5%

BSA were added for 1 hr at room temperature. After

six washings with PBS-T, the plates were developed

with the substrate 5-bromo-4-chloro-3-indolylphos-

phate ⁄ nitroblue tetrazolium (Sigma) for approxi-

mately 5 min until dark blue spots appeared. Then

the reaction was stopped by adding distilled water.

After drying, the numbers of spots were enumerated

by an automated ELISPOT reader (CTL Inc., Cleve-

land, OH, USA).

The total immunoglobulin-secreting cells were

detected with a slight modification of the above-

mentioned procedures. Plates were coated with

70 lL ⁄ well of goat anti-human IgM + IgA + IgG

antibodies (10 lg ⁄ mL diluted in PBS, pH 7.3; Jack-

son ImmunoResearch Laboratories) at 4�C. The

PBMC were used in serial double dilutions from

2.5 · 106 through 4 · 104 cells ⁄ mL (100 lL ⁄ well).

The other steps in the assay were as presented ear-

lier.

Statistical Analysis

Data were presented as mean and standard deviation

(S.D.). Differences between HDCP patients and

healthy pregnant women were analyzed employing

the unpaired student t-test or the non-parametric

Mann–Whitney U-test. A value of P < 0.05 was taken

as statistically significant. Commercially available

GraphPad Prism software (GraphPad Software Inc.,

San Diego, CA, USA) was applied to statistical analysis.

Results

Fetal Monocyte Activation Is Associated with the

Incidence and Severity of HDCP

In this study, cord blood monocytes were obtained

from 46 healthy women delivering term neonates

with normal birth weight after elective cesarean sec-

tion and 60 neonates after term delivery in the pres-

ence of HDCP (20 women with gestational

hypertension, 20 with mild pre-eclampsia and

another 20 with severe pre-eclampsia). The cord

blood monocytes were analyzed for intracellular IL-6

staining by two-color flow cytometry. The gate was

set for both FSC and SSC and included mononuclear

cell population (Fig. 1a). After further gating on

CD14-positive monocytes (Fig. 1b), the two mono-

cyte subpopulations CD14+IL-6) and CD14+IL-6+

were then determined in the CD14 versus interleu-

kin-16 histogram (Fig. 1c).

50 100 150

FSC

P1

(a) (b) (c)P4

P2

P3

Q1-2 Q2-2

Q3-2 Q4-2

CD14 + IL-6-

200 250

5010

015

020

025

0

(× 1,000)

SS

C(×

1,0

00)

50 100 150

FSC FITC-CD14

PE

-IL-

6

102 103 104 105

102

103

104

105

FIT

C-C

D14

CD14+IL-6+

102

103

104

105

200 250(× 1,000)

Fig. 1 Gating strategy for the determination of interleukin-6 (IL-6)-positive monocytes in cord blood. (a) Mononuclear cells were gated based on

forward and side scatter characteristics. After gating on CD14-positive monocytes (b), the two monocyte subpopulations CD14+IL-6) and CD14+IL-

6+ were then determined in the CD14 versus IL-16 histogram (c). P1 gate: mononuclear cell population; P2 gate: CD14high monocyte population;

P3 gate: CD14dim monocyte population; P4: the whole CD14+ monocyte population.

LIU ET AL.



Table I summarizes further clinical data and the

percentage of IL-6-positive monocytes detected in

the cord blood obtained from the patients in these

three groups. In comparison with cord blood cells

obtained from neonates after elective cesarean sec-

tion, a significantly increased percentage of IL-6-

positive monocytes was detected in the cord blood of

women delivering in the presence of HDCP

(P < 0.01) (Table I and Fig. 2). Moreover, the per-

centage of IL-6-positive monocytes was increased as

the disease progressed (P < 0.05) (Fig. 3).

The Increase in Maternal Anti-Fetal Antigen-

Specific ASC Is Associated with the Incidence and

Severity of HDCP

The number of spots observed in the ELISPOT assay

is not a direct reflection of the number of anti-fetal

ASC seeded in a culture well, as proliferation occurs

during the 5-day culture. Therefore, all PBMC sam-

ples were assayed both for total ASC (spots observed

in anti-IgM + IgG + IgA-coated ELISPOT wells) and

anti-fetal antigen-specific ASC (spots observed in

CBMC lysate-coated ELISPOT wells), thereby allow-

ing quantitation of the anti-fetal antigen-specific

ASC as a percentage of total ASC. In the wells as

negative controls, no spots were observed.

The ELISPOT assay specific for anti-fetal immuno-

globulin molecules shows that the percentage of anti-

fetal antigen-specific ASC increased in HDCP women

102 103

FITC-CD14104 105

102

103

PE-I

gG1 10

410

5

102

103

PE-I

L-6 10

410

5

102

103

PE-I

L-6 10

410

5

102

103

PE-I

L-6

104

105

102

103

PE-I

L-6

104

105

102 103

FITC-CD14104 105

102 103

FITC-CD14104 105 102 103

FITC-CD14104 105 102 103

Q3-2

Q1-2

Q4-2

Q2-2

Q3-2

Q1-2

Q4-2

Q2-2

Q3-2

Q1-2

Q4-2

Q2-2

Q3-2

Q1-2

Q4-2

Q2-2

Q3-2

Q1-2

Q4-2

Q2-2

FITC-CD14

92.7%

7.3%6.2%

93.8%95.6%

4.4%0.7%0.1%(a) (b) (c) (d) (e)

99.9% 99.3%

104 105

Fig. 2 Flow cytometric analysis of interleukin-6 (IL-6) expression in CD14-positive cord blood monocytes. Negative staining controls were per-

formed using IgG1 isotype control of irrelevant specificity at the same concentration of the IL-6 monoclonal antibody (mAb) (a). The blood was

obtained from one representative neonate of each group: after normal term pregnancy (b), gestational hypertension (c), mild pre-eclampsia (d)

and severe pre-eclampsia (e). The cord blood mononuclear cells were enriched by density-gradient centrifugation. The cells were stained with fluo-

rescein isothiocyanate-labeled anti-CD14 mAb, and intracellular staining for IL-6 was performed using cell permeabilization reagents. Then the cells

were enumerated by flow cytometric analysis. Percentage of IL-6-positive monocytes was measured among the gated CD14-positive monocyte

population. An increased percentage of IL-6-positive monocytes are detectable in case of gestational hypertension, mild pre-eclampsia and severe

pre-eclampsia.

P

erce

nta

ge

of

IL-6

po

siti

ve m

on

ocy

tes

(%)

* * *

Fig. 3 Percentage of interleukin-6-positive monocytes in cord blood

in normal term pregnant (NP, n = 46), gestational hypertension (GH,

n = 20), mild pre-eclamptic (PE, n = 20) and severe PE (n = 20)

women. *P < 0.05 versus GH group; **P < 0.01 versus GH group. Val-

ues are expressed as mean ± S.D.

NP0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

GH

*

*

*#

Mild PE

Per

cent

age

of a

nti-

feta

lan

tige

n sp

ecif

ic A

SC (

%)

Severe PE

Fig. 4 Percentage of anti-fetal antigen-specific antibody-secreting cells

(ASC) enumerated by enzyme-linked immunosorbent spot-forming cell

assay in normal term pregnant (NP, n = 46), gestational hypertension

(GH, n = 20), mild pre-eclamptic (PE, n = 20) and severe PE (n = 20)

women. Data are represented as the frequency of anti-fetal antigen-

specific ASC as a percentage of the total ASC per million peripheral

blood mononuclear cells. *P < 0.001 compared with normal term

pregnancy; #P < 0.05 compared with gestational hypertension. Values

are expressed as mean ± S.D.




compared with the normal term pregnant subjects

(P < 0.001) (Fig. 4). Moreover, there were statistical

differences between the gestational hypertensive and

severe pre-eclamptic groups (P < 0.05) (Fig. 4).

Discussion

Our data indicate that the fetal monocyte activation

and the increase in maternal anti-fetal antigen-spe-

cific ASC might be related to the incidence and

severity of HDCP. Our findings provide both indirect

and direct evidence in the occurrence of exaggerated

maternal humoral immunity against the fetal anti-

gens in HDCP.

The phenomenon of monocyte activation in the

fetal circulation is common in case of rhesus disease

and some pregnancy-specific diseases such as intra-

uterine growth restriction, pre-eclampsia or HELLP

syndrome, placental abruption and gestational diabe-

tes.10–12 It is a well-known mechanism that the activa-

tion of monocytes occurs through cross-linking of

antibody-loaded cells to monocytes via Fc-receptors,

resulting in the occurrence of IL-6 release.17,18 Previ-

ously published studies further confirmed that the

increased maternal anti-fetal human leukocyte anti-

gen (HLA)-antibodies might be responsible for these,

for they can pass through the placenta and react with

fetal cells (leukocytes and endothelial cells) in the fetal

circulation.10–12 Binding of these antibody-coated cells

to fetal monocytes via Fc-receptors may induce activa-

tion of IL-6 release from these cells. The phenomenon

of monocyte activation in the fetal circulation proba-

bly reflects an increased humoral immune response of

the mother against the fetal antigens.

Our findings are consistent with a previous study,

in which Steinborn et al.10 implicated that the fetal

monocyte activation involved in the pathogenesis of

pre-eclampsia. However, their study recruited only

the patients with pre-eclampsia or HELLP syndrome.

The relationship between the fetal monocyte activa-

tion and other type of HDCP is unknown. Also, it is

not clear whether the fetal monocyte activation

involves in the proceeding of HDCP. In this study,

the study group was divided into three subgroups:

gestational hypertension, mild pre-eclampsia and

severe pre-eclampsia. Our results suggested that the

fetal monocyte activation was related to the severity

of HDCP. The underlying cause remains uncertain.

The possible reason might result from the enhance-

ment of alloimmune stimulation in the pathogenic

process of HDCP.

As previous studies reported, an increasing fetal

traffic between mother and fetus is demonstrated

with advancing gestational age and further enhanced

during pre-eclampsia.19 For example, the fetal nucle-

ated red blood cells and cell-free fetal DNA and

mRNA are increased in maternal blood circulation

from pre-eclamptic patients.20–22 Moreover, the

amounts of these fetal cells are positively correlated

with the severity of pre-eclampsia.23,24 The continu-

ous influx of the alloantigen into maternal circula-

tion stimulates the immune system, leading to the

increase in both memory T cells and B cells. Cha-

iworapongsa et al.25 revealed that patients with pre-

eclampsia had a significantly higher percentage of

CD45RO-positive T-lymphocytes (memory cells) and

a decrease in the percentage of CD45RA-positive T-

lymphocytes (naive cells) than normal pregnant

women. Such an increase in the percentage of

CD45RO-positive memory T cells indicates that

patients with pre-eclampsia have evidence of previ-

ous antigenic exposure. Our recent findings indi-

cated that the percentage of circulating memory B

cells was increased and the capacity of B cells to dif-

ferentiate into antibody-producing cells was also

enhanced in pre-eclamptic women.13 As demon-

strated by the report of Yamamoto et al.,26 B-cell

activation might play a role in the exaggeration of

pre-eclampsia characterized by abnormally high lev-

els of serum autoantibodies, such as the anti-DNA

antibody. Moreover, the degree of B-cell activation

is associated with the severity of HDCP.27

Until now, there is no direct evidence to confirm

the phenomenon that the increased maternal anti-

HLA antibodies did indeed induce the release of IL-6

from fetal monocytes. As these antibodies may

immediately be absorbed by HLA-expressing fetal

cells, the detection of these antibodies in the fetal

circulation was impossible.11 In this way, the pro-

gressively increasing IL-6-positive monocytes in cord

blood indirectly reflected the status of maternal

humoral immune response against the fetal antigens.

To provide more direct data on exaggerated maternal

anti-fetal immunity existing in HDCP, the frequen-

cies of maternal anti-fetal antigen-specific ASC were

evaluated by utilizing the ELISPOT assay. It was

found that the frequencies of maternal anti-fetal

antigen-specific ASC increased in HDCP and were

related to its severity. The result was similar to what

we found in the change of relationship between the

fetal monocyte activation and HDCP. To the best of

our knowledge, this is the first report.

LIU ET AL.



Indeed, the ELISPOT assay is readily adapted as a

laboratory routine for the enumeration of antigen-

specific memory B cells. According to previous

reports,28,29 memory B cells can differentiate into

antigen-specific ASC in vitro under polyclonal activa-

tion like PWM. The frequency of these quiescent B

cells, established between 0.02 and 0.58% IgG-, IgA-

plus IgM- secreting cells, was reported on recent

publications focusing on circulating memory B cells

specific to tetanus toxoid, smallpox, HIV-1 or hepati-

tis B surface Antigens.30–33 In this study, the fre-

quencies of maternal anti-fetal antigen-specific ASC

were 0.37% in the control group, whereas they

were increased in study groups, ranging from 0.67 to

0.94%. It demonstrated that the maternal anti-fetal

humoral immunity existed in normal pregnancy but

enhanced in HDCP. Our results supported the evi-

dence that a humoral immune response of the

mother against paternal HLA-antigens is established

at the end of normal pregnancy. Morin-Papunen

et al.34 evaluated the frequencies of lymphocytotoxic

antibodies, i.e., anti-HLA-ABC antibodies and anti-

HLA-DR antibodies in 66 women at delivery. It

showed that they were, respectively, 18.2 and 9% in

the first pregnancies; 27.3 and 4.5% in the second

pregnancies; and 50 and 27.3% in the multigravious

women (third pregnancy and more). Moreover, Re-

gan et al.35 demonstrated that the incidence of posi-

tive anti-paternal cytotoxic antibodies (APCA) was

related to the duration of pregnancy. It showed that

majority of women who had live births did not

develop APCA until the pregnancy had reached a

gestational age of 28 weeks or more. Doughty and

Gelsthorpe36 also found the incidence of APCA at

delivery to be higher than sera screened at earlier

stages in normal pregnancy. Based on these findings,

we speculated that, in case of HDCP, an excessive

immune response might occur at a very early stage

of pregnancy. However, it needs further investiga-

tions to convince the notion.

In conclusion, the excessive maternal anti-fetal

humoral immunity could be involved in the patho-

genesis and development of HDCP, characterized by

two findings: the progressive increasing IL-6-positive

monocytes in cord blood and the frequencies of

maternal anti-fetal antigen-specific ASC.

Acknowledgments

This work was supported by research grants from

the National Natural Science Foundation of China

(No. 30571710), Graduate’s Innovation Fund of

Huazhong University of Science of Technology

(HF0504107519) and Hubei Province Key Technolo-

gies Program (2006AA301C21).

References

1 Walker JJ: Pre-eclampsia. Lancet 2000; 356:1260–

1265.

2 Duley L: Pre-eclampsia and the hypertensive disorders

of pregnancy. Br Med Bull 2003; 67:161–176.

3 Darmochwal-Kolarz D, Kolarz B, Surdacka A, Rolinski

J, Leszczynska-Gorzelak B, Oleszczuk J: The

expression and concentration of CD40 ligand in

normal pregnancy and pre-eclampsia. J Reprod

Immunol 2009; 79:215–219.

4 Schiessl B: Inflammatory response in preeclampsia.

Mol Aspects Med 2007; 28:210–219.

5 Redman CW, Sargent IL: Latest advances in

understanding preeclampsia. Science 2005; 308:1592–

1594.

6 Saito S, Umekage H, Sakamoto Y, Sakai M, Tanebe K,

Sasaki Y, Morikawa H: Increased T-helper-1-type

immunity and decreased T-helper-2-type immunity in

patients with preeclampsia. Am J Reprod Immunol

1999; 41:297–306.

7 Azizieh F, Raghupathy R, Makhseed M: Maternal

cytokine production patterns in women with pre-

eclampsia. Am J Reprod Immunol 2005; 54:30–37.

8 Sargent IL, Borzychowski AM, Redman CW: NK cells

and pre-eclampsia. J Reprod Immunol 2007; 76:40–44.

9 Toldi G, Svec P, Vasarhelyi B, Meszaros G, Rigo J,

Tulassay T, Treszl A: Decreased number of FoxP3+

regulatory T cells in preeclampsia. Acta Obstet Gynecol

Scand 2008; 87:1229–1233.

10 Steinborn A, Sohn C, Sayehli C, Niederhut A, Schmitt

E, Kaufmann M: Preeclampsia, a pregnancy-specific

disease, is associated with fetal monocyte activation.

Clin Immunol 2001; 100:305–313.

11 Steinborn A, Seidl C, Sayehli C, Sohn C, Seifried E,

Kaufmann M, Schmitt E: Anti-fetal immune

response mechanisms may be involved in the

pathogenesis of placental abruption. Clin Immunol

2004; 110:45–54.

12 Steinborn A, Saran G, Schneider A, Fersis N, Sohn C,

Schmitt E: The presence of gestational diabetes is

associated with increased detection of anti-HLA-class

II antibodies in the maternal circulation. Am J Reprod

Immunol 2006; 56:124–134.

13 Liao AH, Liu LP, Ding WP, Zhang L: Functional

changes of human peripheral B-lymphocytes in pre-

eclampsia. Am J Reprod Immunol 2009; 61:313–321.




14 Sibai BM: Diagnosis and management of gestational

hypertension and preeclampsia. Obstet Gynecol 2003;

102:181–192.

15 Tangye SG, Avery DT, Hodgkin PD: A division-linked

mechanism for the rapid generation of Ig-secreting

cells from human memory B cells. J Immunol 2003;

170:261–269.

16 Rebmann V, Busemann A, Lindemann M, Grosse-

Wilde H: Detection of HLA-G5 secreting cells. Hum

Immunol 2003; 64:1017–1024.

17 Krutmann J, Kirnbauer R, Kock A, Schwarz T, Schopf

E, May LT, Sehgal BP, Luger TA: Cross-linking Fc

receptors on monocytes triggers IL-6 production: role

in anti-CD3- induced T cell activation. J Immunol

1990; 145:1337–1342.

18 Ling ZD, Ziltener HJ, Webb BT, Matheson DS:

Aggregated immunoglobulin and Fc fragment of IgG

induce IL-6 release from human monocytes. Cell

Immunol 1990; 129:95–103.

19 Holzgreve W, Ghezzi F, Di Naro E, Ganshirt D,

Maymon E, Hahn S: Disturbed fetomaternal cell

traffic in preeclampsia. Obstet Gynecol 1998; 91:669–

672.

20 Hahn S, Holzgreve W: Fetal cells and cell-free fetal

DNA in maternal blood: new insights into pre-

eclampsia. Hum Reprod Update 2002; 8:501–508.

21 Jansen MW, Korver-Hakkennes K, van Leenen D,

Visser W, in’t Veld PA, de Groot CJ, Wladimiroff JW:

Significantly higher number of fetal cells in the

maternal circulation of women with pre-eclampsia.

Prenat Diagn 2001; 21:1022–1026.

22 Purwosunu Y, Sekizawa A, Farina A, Wibowo N,

Okazaki S, Nakamura M, Samura O, Fujito N, Okai T:

Cell-free mRNA concentrations of CRH, PLAC1, and

selectin-P are increased in the plasma of pregnant

women with preeclampsia. Prenat Diagn 2007;

27:772–777.

23 Alberry MS, Maddocks DG, Hadi MA, Metawi H,

Hunt LP, Abdel-Fattah SA, Avent ND, Soothill PW:

Quantification of cell free fetal DNA in maternal

plasma in normal pregnancies and in pregnancies

with placental dysfunction. Am J Obstet Gynecol 2009;

200:98.e1–98.e6.

24 Purwosunu Y, Sekizawa A, Koide K, Farina A,

Wibowo N, Wiknjosastro GH, Okazaki S, Chiba H,

Okai T: Cell-Free mRNA concentrations of

plasminogen activator inhibitor-1 and tissue-type

plasminogen activator are increased in the plasma of

pregnant women with preeclampsia. Clin Chem 2007;

53:399–404.

25 Chaiworapongsa T, Gervasi MT, Refuerzo J, Espinoza

J, Yoshimatsu J, Berman S, Romero R: Maternal

lymphocyte subpopulations (CD45RA+ and CD45RO+)

in preeclampsia. Am J Obstet Gynecol 2002; 187:889–

893.

26 Yamamoto T, Yoshimura S, Geshi Y, Sasamori Y, Mori

H, Kobayashi T: Anti-ssDNA and dsDNA antibodies in

preeclampsia. Asia Oceania J Obstet Gynaecol 1994;

20:93–99.

27 el-Roeiy A, Myers SA, Gleicher N: The relationship

between autoantibodies and intrauterine growth

retardation in hypertensive disorders of pregnancy.

Am J Obstet Gynecol 1991; 164(5 Pt 1):1253–1261.

28 Crotty S, Aubert RD, Glidewell J, Ahmed R: Tracking

human antigen-specific memory B cells: a sensitive

and generalized ELISPOT system. J Immunol Methods

2004; 286:111–122.

29 Shokrgozar MA, Sam MR, Amirkhani A, Shokri F:

Frequency analysis of HBsAg-specific B lymphocytes

in high-responder individuals to recombinant

hepatitis B vaccine: comparison of LDA and ELISPOT

assays. Scand J Immunol 2006; 64:536–543.

30 Bernasconi NL, Traggiai E, Lanzavecchia A:

Maintenance of serological memory by polyclonal

activation of human memory B cells. Science 2002;

298:2199–2202.

31 Crotty S, Felgner P, Davies H, Glidewell J, Villarreal

L, Ahmed R: Cutting edge: long-term B cell memory

in humans after smallpox vaccination. J Immunol

2003; 171:4969–4973.

32 Fondere JM, Huguet MF, Yssel H, Baillat V, Reynes J,

van de Perre P, Vendrell JP: Detection of peripheral

HIV-1-specific memory B cells in patients untreated

or receiving highly active antiretroviral therapy. AIDS

2003; 17:2323–2330.

33 Tuaillon E, Tabaa YA, Petitjean G, Huguet MF,

Pajeaux G, Fondere JM, Ponseille B, Ducos J, Blanc P,

Vendrell JP: Detection of memory B lymphocytes

specific to hepatitis B virus (HBV) surface antigen

(HBsAg) from HBsAg-vaccinated or HBV-immunized

subjects by ELISPOT assay. J Immunol Methods 2006;

315:144–152.

34 Morin-Papunen L, Tiilikainen A, Hartikainen-Sorri

AL: Maternal HLA immunization during pregnancy:

presence of anti HLA antibodies in half of

multigravidous women. Med Biol 1984; 62:323–325.

35 Regan L, Braude PR, Hill DP: A prospective study of

the incidence, time of appearance and significance of

anti-paternal lymphocytotoxic antibodies in human

pregnancy. Hum Reprod 1991; 6:294–298.

36 Doughty RW, Gelsthorpe K: A scheme for screening

blood samples to acquire lymphocyte cytotoxic

antisera for tissue typing and results of an initial

screening programme. Transplantation 1971; 12:429–

433.

LIU ET AL.



original article: enhanced maternal anti-fetal immunity contributes to the severity of hypertensive...

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