In many biological systems that involve complexcellular behaviour, cytokines have been observed toexert an important element of control by their abilityto influence cell proliferation, differentiation andmigration. The development of the human placenta ischaracterized by the appearance of large numbers ofchorionic villi in the first trimester of pregnancy.1

The surface of the villi is formed by an outer layer ofsyncytiotrophoblast, a multinucleated zone withoutdistinct cell boundaries, overlying an inner layerof mononuclear cytotrophoblast cells. These villouscytotrophoblast cells are sessile, polarized, epithelialcells resting on a basement membrane and showing evi-dence of proliferative activity. In certain areas of thevilli, cytotrophoblast cells migrate off the basement

membrane and aggregate together to form cyto-trophoblast cell islands or columns. At the placental-uterine interface the cell columns of the anchoringvilli coalesce to form the cytotrophoblast shell. Fromthe shell, individual cytotrophoblast cells break off toinvade deep into the maternal decidua, ultimately dif-ferentiating into placental bed giant cells.2,3 Thus, theprocess of placental implantation exhibits a wide rangeof cellular behaviour which requires strict control. Therole of cytokines in regulating placentation is underactive investigation since the discovery of abundantcytokines at the feto-maternal interface and the demon-stration of appropriate cytokine receptor expression bytrophoblast.4–7

Although several cytokines are believed to beinvolved in human placentation, the exact cellularsources of production for many of them have not beenidentified. In a recent study, we have observed that theunusual population of large granular lymphocytes (nat-ural killer cells) which are present in significant num-bers in decidua at the time of implantation, produces avariety of cytokines.47 This finding lends support to our hypothesis that lymphocyte-derived cytokinescould be involved in the paracrine control of trophoblast development. The purpose of the present study is to determine which cytokines are

364 CYTOKINE, Vol. 7, No. 4 (May), 1995: pp 364–371

SCREENING FOR CYTOKINE mRNA IN HUMANVILLOUS AND EXTRAVILLOUS

TROPHOBLASTS USING THE REVERSE-TRANSCRIPTASE POLYMERASE CHAIN

REACTION (RT-PCR)Ashley King,1 Percy P. Jokhi,1 Steve K. Smith,2 Andrew M. Sharkey,2

Yung Wai Loke1

During the process of placental implantation, sessile villous trophoblast cells migrate fromthe villi into the decidua as isolated motile extravillous trophoblast cells. There is differentialexpression of the epidermal growth factor-receptor (EGF-R) and c-erbB2 proteins on villousand extravillous trophoblast populations. Using monoclonal antibodies to EGF-R and c-erbB2, we have obtained highly purified populations of villous and extravillous trophoblastby fluorescence activated cell sorting. These cells were examined by the reverse transcrip-tase-polymerase chain reaction (RT-PCR) using nested internal primer pairs for the follow-ing cytokines: CSF-1, GM-CSF, TNF-

a, TGF-

b1, IFN-

g, IL-2, LIF and also for LIF-receptor.TNF-

a and TGF-

b1 were present in all trophoblast populations. GM-CSF and CSF-1 wereonly found in some samples, with preferential expression of CSF-1 in villous populations.IFN-

g, IL-2 and LIF mRNA were not found, although all samples contained LIF-receptormRNA. These cytokines (CSF-1, TGF-

b, TNF-

a and GM-CSF) are likely to influence tro-phoblast growth and differentiation in an autocrine manner, since their receptors are alsopresent on trophoblast. These results illustrate a quick and simple method to analyse for thepresence of cytokine and other transcripts in trophoblast subpopulations during early preg-nancy.

From the 1Research Group in Human Reproductive Immunobiology,Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP and 2Department of Obstetricsand Gynaecology, University of Cambridge, Rosie MaternityHospital, Robinson Way, Cambridge CB2 2SW, UK

Correspondence to: Ashley KingReceived 1 August 1994, accepted 10 November 1994© 1995 Academic Press Limited1043-4666/95/040364 +08 $08.00/0

KEY WORDS: Cytokines/Human/RT-PCR/Trophoblast

themselves produced by trophoblast. This would be evi-dence for an additional autocrine route of control. Ofcourse, trophoblast-derived cytokines may in turn,affect uterine cells such as maternal lymphocytes andmacrophages. To investigate this production, it is nec-essary to isolate homogeneous populations of fresh firsttrimester trophoblast cells for analysis. We have previ-ously established that villous cytotrophoblast andextravillous trophoblast can be distinguished by the rec-iprocal expression of the epidermal growth factorreceptor (EGF-R) and c-erbB2 proteins.8 Using mon-oclonal antibodies directed against these two cell sur-face proteins, we have now obtained highly purifiedpreparations of these trophoblast populations usingthe fluorescence-activated cell sorter (FACS). CellularmRNA was extracted and reverse transcribed to cDNA.Expression of CSF-1, GM-CSF, TNF-α, TGF-β1,IFN-γ, IL-2, LIF and LIF-receptor was analysed by thereverse-transcriptase polymerase chain reaction (RT-PCR) using nested internal primer pairs specific forthese cytokines. The results reported here illustrate thatthis method can be used to analyse specific mRNA tran-scripts (such as cytokines) in human first trimester villous and extravillous trophoblast subpopulations.

RESULTS

Highly purified populations of EGF-R1 villouscytotrophoblast and c-erbB21 extravillous trophoblastwere obtained by flow cytometric sorting of freshlyisolated trophoblast. Figure 1A shows first trimesterchorionic villus cells double-stained for the EGF-R andfor the macrophage marker CD14. The fluorescence

gate R2 was used to select EGF-R1 cells while placen-tal macrophages were specifically excluded using thegate R3. c-erbB21 extravillous trophoblast cells wereselected and sorted in a similar way (Fig. 1B). The purityof cell populations obtained using this method wasfound to be in excess of 99%.

Cytokine transcription by first trimester villous andextravillous trophoblast from four individuals (A, B, Cand D) was analysed by RT-PCR using nested internaloligonucleotide primer pairs specific for TNF-α, TGF-β1, GM-CSF, CSF-1, IFN-γ, IL-2, LIF and LIF-recep-tor. These results are shown in Figure 2. Both TNF-α(Fig. 2A) and TGF-b1 (Fig. 2B) were detected in sam-ples of villous cytotrophoblast (lanes 1, 3, 5 and 7) andextravillous trophoblast (lanes 2, 4, 6 and 8) in all cases.For TNF-α, in addition to the major expected productof 666 bp, an additional minor product of ~369 bp wasobtained in all samples (Fig. 2A). Similarly, a minorPCR product of ~220 bp was obtained for TGF-β1 (Fig.2B) in addition to the expected product of 479 bp. Asthese products were present after two roundsamplification using two separate pairs of specificprimers, they are likely to represent splice variants ofTNF-α and TGF-β1.

For GM-CSF, a single band of the expected size (493bp) was observed only in some trophoblast samples,with no consistent differences seen between villous andextravillous trophoblast (Fig. 2C). A different picturewas seen for CSF-1 transcription (Fig. 2D). The CSF-1primers used here are designed to detect three differ-ently spliced forms of CSF-1 mRNA.9 The smallest (170bp) α-form was found to be present in all samples ofvillous cytotrophoblast (lanes 1, 3, 5 and 7), but wasabsent in two out of four extravillous trophoblast samples

Cytokine expression by trophoblast subpopulations / 365

Figure 1. Two colour flow cytometric analysis of isolated first trimester placental cells.

(A) Cells were stained for CD14 and EGF-R. Fluorescence regions R2 and R3 were used to select EGF-R1 CD142 villous tro-phoblast and CD141 EGF-R2 placental macrophages respectively. (B) Cells were stained for CD14 and c-erbB2. Fluorescenceregions R2 and R3 were used to select c-erbB21 CD142 extravillous trophoblast and CD141 c-erbB22 placental macrophagesrespectively.

(lanes 4 and 8) and was difficult to detect in a third (lane6). The larger β and γ forms (1064 and 716 bp respec-tively) were much harder to detect, although this couldbe due to the problem of non-amplification of largerfragments in the presence of a competing smaller frag-ment.

No IFN-γ mRNA expression was seen in either vil-lous or extravillous trophoblast from four individuals(Fig. 2E, lanes 1, 2, 5, 6, 7 and 8). Faint bands of theexpected size were observed in trophoblast populationsfrom one individual (Fig. 2E, lanes 3 and 4), suggestingthat this was an atypical case, or that a very low level ofcontamination by other cell types was being detected.

Similarly, LIF mRNA was undetectable in sevenout of eight trophoblast samples (Fig. 2F, lanes 2–8),suggesting that this cytokine is not normally expressedby the trophoblast populations at this stage of gesta-

tion. However, all samples clearly expressed abundantmRNA for the LIF-receptor, as shown in Figure 2G.

Transcription of IL-2 mRNA was detected in Jurkatcell positive controls (Fig. 2H, lane J), but was absentin both villous and extravillous trophoblast from allsamples (Fig. 2H, lanes 1–8).

DISCUSSION

In the present study, we have screened for mRNAexpression for a variety of cytokines in human firsttrimester trophoblast and have compared the expres-sion between proliferative, sessile villous cytotro-phoblast, and non-mitotic, migratory extravilloustrophoblast. We have previously ascertained thatmonoclonal antibodies against the EGF-R and c-erbB2

366 / King et al. CYTOKINE, Vol. 7, No. 4 (May 1995: 364–371)

Figure 2. Cytokine transcription analysed by RT-PCR.

Expression of mRNA for various cytokines in villous cytotrophoblast (lanes 1, 3, 5 & 7) and extravillous cytotrophoblast (lanes 2, 4, 6 &8) after two rounds of RT-PCR amplification using nested internal primers for (A) TNF-α, (B) TGF-β1, (C) GM-CSF,(D) CSF-1, (E) IFN-γ, (F) LIF, (G) LIF-R, (H) IL-2. J 5 Jurkat derivative cells as positive control for IL-2 mRNA expression;P 5 whole decidua positive control; C 5 yeast tRNA negative control; L 5 123 bp ladder size markers.

IL-2

surface proteins can be used to discriminate betweenvillous and extravillous cytotrophoblast,8 and havetherefore used flow cytometry to obtain highly purifiedpreparations (.99%) of these two trophoblast popula-tions. Cytokine mRNA expression was analysed by RT-PCR amplification using specific nested primer pairs.This method is a quick and simple way to screen for thepresence of specific mRNA transcripts in human tro-phoblast subpopulations. Sorted cells can be obtainedwithin 5 h of the time of tissue collection, and the useof immunofluorescence and cell sorting obviates theneed for culturing cells which can produce in vitro arte-facts. In addition, only small numbers of cells arerequired so this method could be used in future toanalyse the presence of specific mRNA transcripts inplacental biopsies from abnormal pregnancies obtainedby chorionic villous sampling.

In this study, we have detected the presence of sev-eral cytokine transcripts in trophoblast subpopulations.These cytokines (CSF-1, TGF-β, TNF-α and GM-CSF)are likely to influence trophoblast growth and differ-entiation in an autocrine manner, since their receptorsare also present on trophoblast.4,6,7 Production of thesecytokines by trophoblast may also influence otheraspects of implantation by exerting paracrine effects,particularly on maternal cells such as decidual lym-phocytes which might interact with extravillous tro-phoblast in the uterus.

Our data have shown that villous and extravilloustrophoblast express mRNA for TNF-α, which is in accord with an earlier report of demonstration of biologically active TNF-α in amniotic fluid and insupernatant culture media from human placentalexplants.10 We have recently shown that decidual lymphocytes also express TNF-α mRNA,47 so thiscytokine would appear to be derived from both maternal and fetal sources. The possibility that trophoblast is at least one potential target for thiscytokine is suggested by the presence of TNF receptorson these cells.11,12 TNF-α has been reported to inhibitDNA synthesis in human and rat trophoblast celllines,9,10 leading to the hypothesis that this cytokinecould play an important role in limiting the extent oftrophoblast invasion into the uterus.13

The pattern of TGF-β1 mRNA expression in villousand extravillous trophoblast is similar to that of TNF-α. TGF-β1 is also expressed by decidual LGL47 as wellas uterine stromal cells14,15 so again there are placentaland uterine sources for this cytokine in pregnancy.TGF-β1 is known to have a wide range of activities, suchas inhibition of trophoblast proliferation, and inhibitionof trophoblast invasion in in vitro assays.14,16 This anti-invasive effect appears to be mediated by a reductionin Type IV collagenase secretion, and an increase in tissue inhibitor of metalloproteinase (TIMP) pro-duction.16 Furthermore, a wide variety of immuno-

suppressive effects such asinhibition of NK/LAK activity and T cell responses have been ascribed toTGF-βl7,18 all of which could theoretically influence trophoblast behaviour during implantation.

It has been known for some time that human placental-conditioned medium possesses potent colonyforming activity in in vitro bone marrow assays.19,20 Inthe present study we have observed that the α-form ofCSF-1 mRNA was present in all samples of villous tro-phoblast, but was absent or difficult to detect in threeout of four samples of extravillous trophoblast, indi-cating that the ability to produce this cytokine is grad-ually lost as trophoblast migrates into decidua. In anearlier immunohistochemical study, we localized theCSF-1 receptor to human extravillous trophoblast butthese receptors were not detected on villous cyto-trophoblast,4 so it would appear that there is a recipro-cal expression of this cytokine and its receptor on villousand extravillous trophoblast populations. The impor-tance of CSF-1 in pregnancy is underlined by studiesusing osteopetrotic mice which lack a functional CSF-1 gene. Homozygous mutant crosses (op/op 3 op/op)are consistently infertile and this defect is not correctedby systemic administration of CSF-1, demonstrating theabsolute requirement for local CSF-1 production in suc-cessful pregnancy. However, when op/op females aremated with heterozygous (op/+) males, the pregnancieswere partially rescued, suggesting that production ofCSF-1 by op/+ fetuses can compensate for the lack ofmaternal CSF-1.21

Our data on GM-CSF expression is more difficultto interpret. Of the four samples of trophoblastanalysed, two were completely negative while the othertwo cases, one sample of villous cytotrophoblast andanother of extravillous trophoblast, were positive forGM-CSF mRNA. This pattern did not correlate withthe gestational age of the samples, so it is not clear whythere is this inconsistency. We have previously demon-strated that GM-CSF receptors are present on both vil-lous and extravillous trophoblast6 and that CD561

decidual NK cells are an important source of produc-tion of this cytokine.22 These observations suggest thatall trophoblast populations are potentially capable ofresponding to GM-CSF, but that maternally-derivedrather than autocrine production of GM-CSF is morelikely to play a significant role in trophoblast regula-tion. We have shown that GM-CSF causes a modestincrease in 3H-thymidine incorporation by human tro-phoblast in culture.23 This has recently been confirmedin murine trophoblast,24 where it was shown to reflectendoreduplication rather than actual increase in cellnumbers, so it would seem that proliferation may notbe the major response of trophoblast to GM-CSF.However, GM-CSF is also known to enhance the func-tion and increase survival of mature cells by preventingapoptosis,25–27 and preliminary studies in our laboratory

Cytokine expression by trophoblast subpopulations / 367

have shown increased survival of isolated human firsttrimester trophoblast in vitro when cultured withexogenous GM-CSF.

Although previous studies have reported IL-2mRNA expression in syncytiotrophoblast of term andpreterm placentae,28 and IL-2-like protein material inhuman placenta and amnion,29 the presence of IL-2 atthe implantation site is controversial. In the presentstudy, we could detect no IL-2 mRNA expressionin either villous or extravillous trophoblast from nor-mal first trimester pregnancies. This absence of IL-2expression, particularly by invasive extravillous tro-phoblast within the uterus, is likely to be highlysignificant, since IL-2 transforms uterine CD56bright

CD162 NK cells into potent lymphokine activated killer(LAK) cells which are capable of lysing first trimestertrophoblast in vitro.30 Similarly, IFN-γ is another cyto-kine with potent effects on both immune and non-immune cells. IFN-γ receptors are expressed by humantrophoblast5 and we have previously demonstrated thattreatment of first trimester trophoblast with IFN-γ par-tially protects these cells from lysis by IL-2 transformeddecidual NK cells, and that this may be due to the smallupregulation of surface HLA Class I antigens seen afterIFN-γ treatment.31 However, our data suggest that anautocrine action of IFN-γ on first trimester trophoblastis unlikely, since mRNA for this cytokine was notdetected in the majority of either villous or extravilloustrophoblast samples. It is possible, however, that IL-2and IFN-γ may be produced in pathological conditions.

Trophoblast expression of mRNA for LIF and LIFreceptor was also investigated because LIF has recentlybeen shown to play a critical role in blastocyst implan-tation. LIF mRNA expression in uterine endometrialglands occurs just prior to implantation in pregnantmice,32 and experiments using transgenic ‘knockout’mice have shown that females lacking a functional LIF gene are infertile, but that their blastocysts them-selves are viable, and can implant and develop to termwhen transferred to wild-type pseudopregnant recipients.33 We have recently demonstrated LIFmRNA expression in both maternal uterine NK cells and T cells.47 Our present study indicates that LIFreceptors are indeed expressed by trophoblast, but not LIF itself, which suggests that as in mice, the source of LIF during pregnancy is likely to be maternally derived.

In conclusion, we have shown that using this method,mRNA can be detected for various cytokines. With theexception of CSF-1, no differences could be detectedbetween villous and extravillous trophoblast popula-tions. Immunohistological studies have demonstratedthe presence of many of these cytokine proteins in troph-oblast,14,15,34–36 indicating that this technique will providea valid screening procedure to examine for the presenceof specific mRNA in trophoblast subpopulations.

MATERIALS AND METHODS

Samples and cell lines

Trophoblast. First trimester (6–12 weeks gestation)tissue from four individuals was obtained from elective ter-minations of pregnancy at Addenbrooke’s Hospital(Cambridge, UK). Chorionic villous tissue was macroscopi-cally separated from decidual tissue, and washed in HAMSF12 medium (ICN Flow, High Wycombe, UK). The chorionicvilli were then finely minced between two scalpel blades andincubated for 10 min at 37ºC in 0.25% trypsin 0.02% EDTA(Difco, Detroit, Michigan) to achieve enzymatic disaggrega-tion of cells. The resultant cell suspension was then filteredthrough muslin and centrifuged at 400 3 g for 5 min. The cellsuspension was resuspended in HAMS F12, layered ontoLymphoprep (ICN Flow) and spun at 600 3 g for 20 min. Thecells at the interface were removed, resuspended in HAMSF12 1 20% FCS and filtered through a 75 µm sieve(Gallenkamp) to remove multinucleated syncytial knots fromthe cell suspension. The cells were then washed, and resus-pended in phosphate buffered saline with 2% FCS (PBS/FCS)at 4ºC for immunofluorescent labelling and flow cytometricsorting of trophoblast subsets.

Jurkat derivative (CRL 8163) cells were used as a posi-tive control for IL-2 mRNA expression, and were obtainedfrom the American Tissue Culture Collection and maintainedin RPMI 1640 (ICN Flow) with 10% FCS, supplemented withantibiotics and 2 mM L-Glutamine.

Fluorescence-activated cell sorting (FACS) oftrophoblast subsets

Enzymically-disaggregated trophoblast cells in suspen-sion were incubated for 30 min at 4ºC with a 1 in 10 dilutionof mouse monoclonal antibody (mAb) to either the EGF-R(Ab-1, Oncogene Science) to label villous cytotrophoblast, orto c-erb B2 (Ab-5, Oncogene Science) to label extravilloustrophoblast. The cells were then washed twice in PBS/FCS,and incubated for 30 min at 4ºC with a 1 in 50 dilution of sec-ondary fluorochrome-conjugated antibody (rabbit anti-mouse IgG-FITC F(ab9)2 fragments) (Serotec). Cells werethen washed twice, incubated with purified mouse IgG toquench any residual FITC-conjugated rabbit anti- mouse sec-ondary Ab, and then double-stained with a phycoerythrin(PE)-conjugated mouse mAb to CD14 (Becton Dickinson)to label placental macrophages. This was done to ensure thatthere was no contamination of trophoblast samples with pla-cental macrophages during cell sorting. Purified populationsof first trimester EGF-R1 c-erbB22 CD142 villous cytotro-phoblast and c-erbB21 EGF-R2, CD 142 extravillous tro-phoblast were obtained by flow cytometric sorting ofimmunolabelled cell preparations using a Becton DickinsonFACStar flow cytometer. 100 000 cells of each subset wereobtained, and the purity was determined to be in excess of99% by subsequent FACS analysis. The sorted cells were then spun down at 400 3 g for 5 min and the cell pellets snap frozen in liquid nitrogen and stored at 270ºC, untilmRNA extraction. Cells were frozen down within 5 h of tis-sue collection.

368 / King et al. CYTOKINE, Vol. 7, No. 4 (May 1995: 364–371)

Cytokine expression by trophoblast subpopulations / 369

TAB

LE

1.P

rim

ers

used

for

RT-

PC

R

Spec

ific

ity

Pri

mer

Sequ

ence

59

→ 3

9Fr

agm

ent s

ize

(bp)

Posi

tion

on

cDN

AR

efer

ence

(cD

NA

sequ

ence

)

TN

F-α

Ext

erna

l (59

end

)A

TG

AG

CA

CT

GA

AA

GC

AT

G86

–103

Ext

erna

l (39

end

)T

CA

CA

GG

GC

AA

TG

AT

CC

C70

278

4–77

0In

tern

al (

59 e

nd)

AT

CC

GG

GA

CG

TG

GA

GC

TG

104–

121

Inte

rnal

(39

end)

AA

AG

TA

GA

CC

TG

CC

CA

GA

C66

676

9–75

1W

ang

et a

l.,19

8539

IFN

-γE

xter

nal (

59en

d)A

TG

AA

AT

AT

AC

AA

GT

TA

TA

TC

129–

149

Ext

erna

l (39

end)

AA

AT

TC

AA

AT

AT

TG

TA

GG

CA

535

663–

644

Inte

rnal

(59

end)

TT

GG

CT

TT

CA

GC

TC

TG

CA

T15

0–17

0In

tern

al (

39en

d)G

GA

CA

AC

CA

TT

AC

TG

GG

AT

G49

464

3–62

4N

ishi

et a

l., 1

98540

TG

F-β

1E

xter

nal (

59en

d)A

AC

AC

AT

CA

GA

GC

TC

CG

AG

AA

1248

–126

9E

xter

nal (

39en

d)G

TC

AA

TG

TA

CA

GC

TG

CC

GC

AC

500

1748

–172

7In

tern

al (

59en

d)G

CG

GT

AC

CT

GA

AC

CC

GT

GT

T12

70–1

289

Inte

rnal

(39

end)

GT

CA

AT

GT

AC

AG

CT

GC

CG

CA

C*

479

1748

–172

7D

eryn

ck e

t al.,

1985

41

CSF

-1E

xter

nal (

59en

d)C

TC

CT

TG

AC

AA

GG

AC

TG

GA

620–

638

Ext

erna

l (39

end)

CT

CT

GG

TT

GC

TC

CA

AG

GG

A24

9-α/

1143

-β/7

95-γ

1762

–174

4 In

tern

al (

59en

d)T

GC

TG

AA

TG

CT

CC

AG

CC

AA

670–

688

Inte

rnal

(39

end)

TC

TG

AG

GC

TC

TT

GA

TG

GC

T17

0-α/

1064

-β/7

16-γ

1733

–171

5W

ong

et a

l., 1

98742

GM

-CSF

Ext

erna

l (59

end)

AT

GT

GA

AT

GC

CA

TC

CA

GG

AG

GC

103–

124

Ext

erna

l (39

end)

CC

CA

TT

CT

TC

TG

CC

AT

GC

CT

GT

515

617–

596

Inte

rnal

(59

end)

CC

GG

CG

TC

TC

CT

GA

AC

CT

GA

125–

144

Inte

rnal

(39

end)

CC

CA

TT

CT

TC

TG

CC

AT

GC

CT

GT

*49

361

7–59

6W

ong

et a

l., 1

98543

Il-2

Ext

erna

l (59

end)

AA

CT

CC

TG

TC

TT

GC

AT

TG

CA

61–8

0E

xter

nal (

39en

d)G

TG

TT

GA

GA

TG

AT

GC

TT

GA

C44

150

1–48

1 In

tern

al (

39en

d)C

TA

AG

TC

TT

GC

AC

TT

GT

CA

C81

–100

In

tern

al (

39en

d)G

GT

AA

TC

CA

TC

TG

TT

CA

GA

A39

647

6–45

7Ta

nigu

chi e

t al.,

198

344

LIF

Ext

erna

l (59

end)

AG

AT

CA

GG

AG

CC

AA

CT

GG

CA

187–

206

Ext

erna

l (39

end)

AC

CC

AA

CT

CC

TG

AG

AT

CC

CT

517

703–

684

Inte

rnal

(59

end)

CA

GC

TC

AA

TG

GC

AG

TG

CC

AA

207–

226

Inte

rnal

(39

end)

GT

TC

AC

AG

CA

CA

CT

TC

AA

GA

C47

768

1–66

1M

orea

u et

al.,

198

845

LIF

-rec

epto

rE

xter

nal (

59en

d)G

AA

AA

CT

GT

AA

AG

CA

TT

AC

A28

07–2

826

Ext

erna

l (39

end)

AG

AG

TC

TG

GA

GA

CA

CT

AA

504

3310

–329

3 In

tern

al (

59en

d)C

AA

AA

GA

GT

GT

CT

GT

GA

G28

31–2

848

Inte

rnal

(39

end)

CC

AT

GT

AT

TT

AC

AT

TG

GC

459

3289

–327

2G

eari

ng e

t al.,

199

146

* In

dica

tes

a he

mi-

nest

ed p

rim

er.

Preparation of RNA and reverse-transcription tocDNA

RNA was prepared from the frozen cell pellets by themethod of Chomczynski and Sacchi37 and reverse transcribedas described previously.38 Briefly, the cell pellets were homog-enized in a buffer containing guanidinium thiocyanate (GibcoBRL, UK) and total RNA purified by acid phenol extractionand ethanol precipitation. Fifty µg of yeast tRNA (Gibco)were added to each sample as a carrier. cDNA was synthe-sized using AMV reverse transcriptase (super RT, HTBiotechnology, Cambridge, UK).

Oligonucleotide primers

Oligonucleotide primers for GM-CSF, CSF-1, TNF-α,TGF-β1, IFN-γ, IL-2, LIF and LIF-receptor were synthesizedon a Cruachem PS250 DNA synthesizer. Primer sequenceswere designed from published nucleotide sequences (See Table1), such that amplification of any contaminating genomic DNAwould result in a differently sized product from the cDNAspecies. Two pairs of primers were designed for each cytokine—an outer pair for first round amplification, and a nested inter-nal pair which was used in a second round of PCR amplificationto confirm the specificity of the PCR products.

RT-PCR amplification

PCR amplification of the cDNA preparations was per-formed as previously described38 with a Hybaid OmnigeneDNA thermal cycler in a final volume of 30 µl using 1 U ofTaq DNA polymerase (SuperTaq, HT Biotechnology,Cambridge, UK) and 1 µM of each of the pair of externalprimers (see Table 1) in the manufacturer’s recommendedbuffer. The following cycle profile was used: 30s at 95ºC, 30sat XºC, 30s at 72ºC for 30 cycles, where X is the annealing tem-perature for each pair of cytokine primers:

External (ºC) Internal (ºC)

TNF-α 49 54 IFN-γ 48 54 TGF-β1 61 61 CSF-1 55 54 GM-CSF 62 62 IL-2 57 57 LIF 60 60 LIF-R 48 48

Samples of cDNA reverse transcribed from mRNAextracted from whole decidua were used as positive controls,while reaction mixtures containing only yeast tRNA carrierwere used as negative controls. After the first round of PCRamplification, 1 µl of the products was transferred to a fresh30 µl reaction mix containing the inner pair of primers andreamplified for 25 cycles. Twenty µl of the products were thenanalysed by 2% agarose gel electrophoresis in the presenceof 0.5 µg/ml ethidium bromide, and their identity wasconfirmed by non-radioactive sequencing.

Acknowledgements

We would like to thank our obstetric colleagues andstaff at Addenbrooke’s Hospital for collecting the pla-cental material, Steve Charnock-Jones for advice, EvaRainbow-Hills, Marie Mack and Kate Day for techni-cal assistance, and Jenny Connor for typing the manu-script.

This work was supported by the Medical ResearchCouncil and the Special Programme of Research,Development and Research Training in HumanReproduction, World Health Organization. PPJ is arecipient of a Wellcome Prize Studentship for theMB/PhD programme at the School of ClinicalMedicine, University of Cambridge. AK is in receipt ofthe Meres Senior Studentship for Medical Research atSt. John’s College, Cambridge.

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