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G anulocy e supe oxide anion p oduc ion and egula ion by plasma ac o s in no mal
and p eeclamp ic p egnancy
Rudol Lampéa*, Sándo Szűcsb, Róza Ádányb and Robe Pókaa
Depa men s o Obs e ics and Gynecology a and P e en i e Medicine, Facul y o Public
Heal h b, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Hunga y
*Add ess o co espondence:
Rudol Lampé M.D.
Depa men o Obs e ics and Gynecology
Uni e si y o Deb ecen Medical and Heal h Science Cen e
Nagye dei k . 98., Deb ecen, 4012-Hunga y
Phone: +36 52 417144; Fax: +36 52 417171;
e-mail: [email p o ec ed]
*Manusc ip
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Abs ac
Da a on he espi a o y bu s ac i i y o g anulocy es om heal hy and p eeclamp ic women
ha e emained con adic o y. To in es iga e he ole o eac i e oxygen species in he e iology
o p eeclampsia we measu ed supe oxide anion gene a ion by g anulocy es om non-
p egnan , heal hy, and p eeclamp ic women. We also examined he ecip ocal e ec s o hea -
inac i a ed and non-inac i a ed plasma on supe oxide p oduc ion. Supe oxide gene a ion was
measu ed by e icy och ome-c educ ion. Supe oxide p oduc ion induced by ei he pho bol-
12.13-dibu i a e o N- o myl-me hionyl-leucyl-phenylalanine was signi ican ly dec eased in
g anulocy es om no mal p egnan women compa ed wi h non-p egnan and p eeclamp ic
women. The pho bol-12.13-dibu i a e-induced supe oxide gene a ion by g anulocy es om
non-p egnan and p eeclamp ic women was signi ican ly inhibi ed by plasma om heal hy
p egnan women. The N- o myl-me hionyl-leucyl-phenylalanine-s imula ed supe oxide
p oduc ion by g anulocy es om non-p egnan and p eeclamp ic women was supp essed only
by non-inac i a ed plasma, no hea -inac i a ed plasma om heal hy p egnan women. Plasma
om p eeclamp ic women did no in luence he pho bol-12.13-dibu i a e- and N- o myl-
me hionyl-leucyl-phenylalanine-induced supe oxide p oduc ion by con ol g anulocy es. The
pho bol-12.13-dibu i a e-induced supe oxide gene a ion by g anulocy es om heal hy
p egnan women was signi ican ly inc eased by he e ec o plasma om non-p egnan and
p eeclamp ic women, bu when s imula ing wi h N- o myl-me hionyl-leucyl-phenylalanine
only non-inac i a ed plasma caused he same enhancemen . These da a indica e ha educed
supe oxide gene a ion in no mal p egnancy may be caused by ma e nal immunosupp essi e
ac o s p esen in plasma. The ailu e o educe supe oxide p oduc ion in p eeclampsia may be
pa ly esponsible o he endo helial dys unc ion cha ac e is ic o ha condi ion.
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Key wo ds: p eeclampsia; no mal p egnancy; g anulocy es; supe oxide anion p oduc ion;
oxida i e s ess; plasma ac o s
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1. In oduc ion
P eeclampsia is a p egnancy-speci ic mul isys em diso de ha de elops in 3–10% o
human p egnancies and is he leading cause o ma e nal and pe ina al mo bidi y and
mo ali y, especially in de eloped coun ies (Da ey and MacGilli ay, 1988). I is
cha ac e ized by an abno mal ascula esponse o placen a ion, esul ing in mild o se e e
ma e nal hype ension, p o einu ia, enhanced pla ele agg ega ion and ac i a ion o he blood
coagula ion sys em (Sibai e al., 2005). In addi ion o gene ic ac o s ma e nal endo helial cell
damage and dys unc ion ha e been p oposed o be implica ed in he pa hogenesis o he
disease (Mellembakken e al., 2002). Al hough he exac mechanism is no ye known, an
excessi e ma e nal in lamma o y esponse o p egnancy and he ac i a ion o g anulocy es by
placen ally eleased ci cula o y ac o s ha e been sugges ed o con ibu e o endo helial
damage in p eeclampsia (Aly e al., 2004). In e ac ion o hese ac o s wi h g anulocy es may
induce he p oduc ion o supe oxide anions (O2.-) and ela ed eac i e oxygen species (ROS)
(Hubel, 1999). Supe oxide anions gene a ed by ac i a ed g anulocy es may ini ia e oxida i e
s ess, lipid pe oxida ion and endo helial cell lysis (G a acós, 2000).
S udies on O2.- p oduc ion by g anulocy es om no mal and p eeclamp ic women ha e
also p o ided con lic ing esul s. The da a published by Tsukimo i e al. (1993) demons a ed
ha N- o myl-me hionyl-leucyl-phenylalanine (FMLP)-s imula ed O2.- gene a ion by
g anulocy es was inc eased in women wi h p eeclampsia compa ed wi h heal hy p egnan
women, and no signi ican di e ence was ound be ween O2.- p oduc ion by g anulocy es
om no mal p egnan women and ha by g anulocy es om heal hy non-p egnan women.
Sel a aj e al. (1982) showed a highe g anulocy e O2.- le el in no mal p egnancy compa ed
wi h non-p egnan con ols. Sacks e al. (1998) measu ed an inc easing oxygen adical
p oduc ion o g anulocy es in no mal p egnancy and p eeclampsia, bu we e unable o
demons a e a signi ican di e ence be ween hese g oups. In con as , C ocke e al. (1999;
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2000) epo ed ha he FMLP-induced g anulocy e O2.- gene a ion was signi ican ly educed
in heal hy p egnan women compa ed wi h non-p egnan con ols, and hey ound no
signi ican di e ence be ween O2.- gene a ion by g anulocy es om pa ien s wi h
p eeclampsia and g anulocy es om heal hy non-p egnan women. C ouch e al. (1995) and
o he s ha e shown signi ican ly lowe g anulocy e O2.- p oduc ion in heal hy p egnan women
compa ed wi h non p egnan con ols (Mille and Russell, 1986).
Se e al ci cula o y ac o s can in luence O2.- p oduc ion in no mal and p eeclamp ic
p egnancies (Redman and Sa gen , 2003). The e a e supp essi e e ec s in he ma e nal
immune sys em in no mal p egnancy; he e o e, i is easonable o suppose ha
immunosupp essi e ac o s may also be p esen in he ci cula ion o he mo he (Viganò e al.,
2007). Fo his eason he educed O2.- gene a ion in heal hy p egnancy and no mal O2.-
p oduc ion in women wi h p eeclampsia may be due o he p esence and absence o hese
ac o s in he plasma o no mal and p eeclamp ic women, espec i ely. The pu pose o ou
cu en s udy was o es his hypo hesis. The e o e, we aimed o examine ecip ocally
whe he plasma samples om heal hy non-p egnan , no mal and p eeclamp ic women could
in luence he pho bol-12,13-dibu i a e- (PDBu) and FMLP-induced O2.- p oduc ion by
g anulocy es om non-p egnan con ols, and heal hy and p eeclamp ic women. Fu he mo e,
he O2.- p oduc ion by g anulocy es om heal hy non-p egnan , no mal and p eeclamp ic
women was also measu ed using he abo e-men ioned s imula ing agen s.
2. Ma e ials and me hods
2.1. S udy popula ion
A e in o med consen and he app o al o he Ins i u ional E hics Commi ee,
pe iphe al blood was collec ed om 31 no mal and 39 p eeclamp ic women in hei hi d
imes e o p egnancy. Blood samplings we e pe o med in ges a ional weeks 26–38. A

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g oup o 35 age-ma ched non-p egnan women se ed as con ols in his s udy. P eeclampsia
was de ined as de elopmen o hype ension a e he 20 h week o p egnancy ( 140/90 mm
Hg measu ed in wo consecu i e occasions 6 hou s apa ) and p o einu ia o highe han 300
mg/day. The p eeclamp ic g oup included 9 mildly and 30 mode a ely ill pa ien s wi h blood
p essu e o 140/90–149/99 mm Hg and 150/100–159/109 mm Hg, espec i ely (ACOG
Commi ee on Obs e ic P ac ice, 2002). All o he pa ien s wi h p eeclampsia we e no on
any medica ion, wi hou a his o y o diabe es melli us and wi h an absence o majo medical
disease o su gical in e en ion.
2.2 Sepa a ion o g anulocy es om pe iphe al blood
Pe iphe al blood was collec ed in acu aine es ubes con aining EDTA (Bec on-
Dickinson, Cedex, F ance). Blood samples we e laye ed on he op o a Ficoll solu ion (1.077
g/mL) and he supe na an con aining he leukocy es was emo ed a e sedimen a ion o
e y h ocy es a 1 g o 60 min a oom empe a u e. The leukocy e- ich plasma was laye ed on
op o a discon inuous Ficoll g adien (1.077 and 1.119 g/mL) and cen i uged a 350 g a
20C o 30 min. G anulocy es sedimen ed a he in e ace o he Ficoll laye s we e collec ed
and washed wice wi h Hanks’ solu ion, pH 7.4 a 20C (English and Ande sen, 1974). Cell
iabili y checked by he ypan blue exclusion es was ound o be 98%. The pu i y o he
g anulocy e suspensions a ied be ween 94 and 98% as e ealed by mic oscopic
examina ions. Red blood cells we e no emo ed by hypo onic lysis since he e y h ocy e
con amina ion in he g anulocy e suspensions was negligible.
2.3. Measu emen o supe oxide anion p oduc ion
Supe oxide anion elease was measu ed by supe oxide dismu ase-inhibi able (SOD,
om bo ine e y h ocy es, 4200 U/mg p o ein) educ ion o e icy och ome-c (Babio e al.,
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1975). G anulocy es (3x105) we e incuba ed in Hanks’ solu ion (pH 7.4) wi h pho bol-12,13-
dibu y a e (PDBu) o n- o myl-me hionyl-leucyl-phenylalanine (FMLP) a 37C o 15 min.
The o al assay olume was 0.5 mL. The inal concen a ions o SOD, e icy och ome-c,
PDBu, and FMLP we e 100 U/mL, 50 mol/L, 100 nmol/L, and 1 mol/L, espec i ely. The
change in abso bance was measu ed spec opho ome ically a 550 nm wi h a double beam
Shimadzu UV-160A spec opho ome e (Shimadzu Seisakusho L d., Kyo o, Japan) a oom
empe a u e. The amoun o supe oxide anion sec e ed in o he medium was calcula ed on he
basis o he mola ex inc ion coe icien o educed cy och ome-c 2.1x104 M-1cm-1 (Pick and
Keisa i, 1981).
2.4. E ec o plasma samples on supe oxide anion p oduc ion by g anulocy es om non-
p egnan , heal hy p egnan , and p eeclamp ic women
Plasma ac ions we e isola ed om pe iphe al blood o heal hy non-p egnan and
no mal p egnan women as well as pa ien s wi h mode a e p eeclampsia by cen i uga ion a
800 g a 20C o 10 min and hen he plasma samples we e di ided in o wo pa s. To check
he sugges ed e ec o he complemen sys em on he supe oxide p oduc ion o g anulocy es
(Rossi, 1986) inac i a ed and non-inac i a ed plasma samples we e used. Hal o each
ac ion was hea ed o 56C o 30 min, while he o he po ion was no inac i a ed.
Subsequen ly, he indi idual plasma p epa a ions we e no pooled. E e y expe imen was
pe o med wi h inac i a ed and non-inac i a ed plasma samples simul aneously. G anulocy es
(3x106) om heal hy non-p egnan women we e incuba ed wi h plasma samples (1.5 ml) om
no mal and p eeclamp ic women. G anulocy es (3x106) om no mal p egnan women we e
ea ed wi h plasma p epa a ions (1.5 ml) om heal hy non-p egnan and p eeclamp ic
women. G anulocy es (3x106) om p eeclamp ic women we e incuba ed wi h plasma
ac ions (1.5 ml) o non-p egnan and heal hy p egnan women. In o de o examine he
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possibili y o s imula ion o supp ession o O2.- p oduc ion due o an immune eac ion o he
cells wi h o eign plasma ac o s, g anulocy es (3x106) om non-p egnan , heal hy p egnan ,
and p eeclamp ic women we e also ea ed wi h au ologous and he e ologous plasma samples
(1.5 ml) o non-p egnan , no mal, and p eeclamp ic women espec i ely. Following
incuba ion o he cells a 37C o 1 h, g anulocy es we e washed wi h Hanks’ solu ion and
O2.- p oduc ion was measu ed as desc ibed abo e.
2.5. S a is ical analysis
The esul s a e p esen ed as means (SD). The dis ibu ion o da a, checked by he
Kolmogo o –Smi no es , was no mal. Di e ences among he clinical pa ame e s o he
s udy popula ion, supe oxide anion p oduc ion by g anulocy es om non-p egnan con ols,
no mal p egnan women, and p eeclamp ic women as well as O2.- gene a ion by g anulocy es
om non-p egnan , heal hy p egnan , and p eeclamp ic women ea ed wi h app op ia e
plasma samples we e de e mined by one-way analysis o a iance (ANOVA) using he
Newman–Keuls pos -hoc es . The da a in Figs. 1-3 ep esen he mean alues (SD) ob ained
om independen expe imen s wi h g anulocy es and plasma samples isola ed om six
indi idual women pe g oup. Values o p0.05 we e conside ed o be s a is ically signi ican .
3. Resul s
The clinical pa ame e s o he s udy popula ion a e p esen ed in Table 1. The e we e
signi ican di e ences in sys olic and dias olic blood p essu es (p<0.001), body mass index
(BMI, p<0.05), ges a ional age a deli e y (p<0.01), p o einu ia a he ime o blood sampling
(p<0.001), and bi h weigh (p<0.01) be ween no mal p egnan and p eeclamp ic women.
3.1. Supe oxide anion p oduc ion by g anulocy es
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Supe oxide anion p oduc ion by g anulocy es om heal hy non-p egnan , no mal
p egnan , and p eeclamp ic women in esponse o PDBu and FMLP s imula ion is illus a ed
in ou p e ious wo k (Lampé e al., 2008). As shown he e, using he wo s imula o agen s
(PDBu and FMLP), g anulocy es om no mal p egnan women demons a ed signi ican ly
dec eased O2.- gene a ion compa ed wi h non-p egnan and p eeclamp ic women. The e was
no signi ican di e ence in O2.- p oduc ion by g anulocy es om non-p egnan con ols and
ha by g anulocy es om pa ien s wi h p eeclampsia. G anulocy es om p eeclamp ic
women eleased a signi ican ly g ea e amoun o O2.- compa ed wi h no mal p egnan
women.
3.2. E ec s o plasma samples on supe oxide anion p oduc ion
The e ec s o plasma ac ions om no mal p egnan and p eeclamp ic women on O2.-
p oduc ion by g anulocy es om heal hy non-p egnan women a e illus a ed in Fig. 1A and
1B. As shown in Fig. 1A, bo h inac i a ed plasma (IP) and non-inac i a ed plasma (NIP) om
heal hy p egnan women signi ican ly inhibi ed he PDBu-induced O2.- gene a ion by
g anulocy es om non-p egnan con ols compa ed wi h O2.- gene a ion by he cells ea ed
wi h au ologous and he e ologous IP and NIP om heal hy non-p egnan women as well as
p eeclamp ic women. The e we e no signi ican di e ences in PDBu-s imula ed O2.-
gene a ion by g anulocy es om non-p egnan con ols a e incuba ion o he cells wi h
au ologous and he e ologous plasma om non-p egnan women as well as plasma om
p eeclamp ic women. As depic ed in Fig. 1B, he FMLP-s imula ed O2.- p oduc ion by
g anulocy es om non-p egnan con ols was no in luenced by IP om heal hy p egnan and
p eeclamp ic and he e ologous non-p egnan women. Howe e , incuba ion o he cells wi h
NIP om heal hy p egnan women esul ed in a signi ican inhibi ion o O2.- gene a ion
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Re e ences
ACOG Commi ee on Obs e ic P ac ice. 2002. ACOG p ac ice bulle in. Diagnosis and
managemen o p eeclampsia and eclampsia. Numbe 33, Janua y 2002. Ame ican
College o Obs e icians and Gynecologis s. In . J. Gynecol. Obs e . 77, 67–75.
Aly, A.S., Khandelwal, M., Zhao, J., Mehme , A.H., Sammel, M.D., Pa y, S. 2004.
Neu ophils a e s imula ed by syncy io ophoblas mic o illous memb anes o gene a e
supe oxide adicals in women wi h p eeclampsia. Am. J. Obs e . Gynecol. 190, 252-
258.
Babio , B.M., Cu nu e, J.T., Kipnes, R.S. 1975. Biological de ense mechanisms. E idence
o he pa icipa ion o supe oxide in bac e ial killing by xan hine oxidase. J. Lab. Clin.
Med. 85, 235-244.
C ocke , I.P., Wellings, R.P., Fle che , J., Bake , P.N. 1999. Neu ophil unc ion in women
wi h p e-eclampsia. B . J. Obs e . Gynaecol. 106, 822-288.
C ocke , I.P., Bake , P.N., Fle che , J. 2000. Neu ophil unc ion in p egnancy and heuma oid
a h i is. Ann. Rheum. Dis. 59, 555-564.
C ouch, S.P.M., C ocke , I.P., Fle che , J. 1995. The e ec o p egnancy on
polymo phonuclea leukocy e unc ion. J. Immunol. 155, 5436-5443.
Da ey, D.A., MacGilli ay, I. 1988. The classi ica ion and de ini ion o he hype ensi e
diso de s o p egnancy. Am. J. Obs e . Gynecol. 158, 892–898.
English, D., Ande sen, B,R. 1974. Single-s ep sepa a ion o ed blood cells. G anulocy es and
mononuclea leukocy es on discon inuous densi y g adien s o Ficoll-Hypaque. J.
Immunol. Me hods. 5, 249-252.
Goel, G., Makka , H.P.S., F ancis, G., Becke , K. 2007. Pho bol es e s, s uc u e, biological
ac i i y, and oxici y in animals. In . J. Toxicol. 26, 279-288.

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Figu e Legends
Figu e 1.
E ec o plasma samples on PDBu- (A) and FMLP-s imula ed (B) supe oxide anion
p oduc ion by g anulocy es om non-p egnan con ols. G anulocy es we e incuba ed wi h
inac i a ed (IP) and non-inac i a ed (NIP) plasma om heal hy and p eeclamp ic women as
well as wi h au ologous and he e ologous non-p egnan plasma. Mean alues (SD) ob ained
om independen expe imen s wi h g anulocy es and plasma samples isola ed om six
indi idual women/g oup a e demons a ed. Signi ican di e ences a e indica ed as ollows:
***p < 0.001 au ologous IP e sus heal hy p egnan IP; ##p < 0.01, ###p < 0.001 au ologous
NIP e sus NIP om heal hy p egnan women.
Figu e 2.
E ec o plasma samples on PDBu- (A) and FMLP-induced (B) supe oxide anion p oduc ion
by g anulocy es om heal hy p egnan women. G anulocy es we e incuba ed wi h inac i a ed
(IP) and non-inac i a ed (NIP) plasma samples om non-p egnan con ols and p eeclamp ic
women as well as wi h au ologous and he e ologous plasma om no mal p egnan subjec s.
Mean alues (SD) ob ained om independen expe imen s wi h g anulocy es and plasma
samples isola ed om six indi idual women/g oup a e p esen ed. Signi ican di e ences a e
indica ed as ollows: ***p < 0.01 au ologous IP e sus heal hy non-p egnan IP and
p eeclamp ic IP; ###p < 0.001 au ologous NIP e sus NIP om heal hy non-p egnan and
p eeclamp ic women.
Figu e 3.
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E ec o plasma samples on PDBu- (A) and FMLP-s imula ed (B) supe oxide anion
p oduc ion by g anulocy es om p eeclamp ic women. G anulocy es we e incuba ed wi h
inac i a ed (IP) and non-inac i a ed (NIP) plasma samples om non-p egnan con ols and
heal hy p egnan subjec s as well as wi h au ologous and he e ologous plasma om
p eeclamp ic women. Mean alues (SD) ob ained om independen expe imen s wi h
g anulocy es and plasma samples isola ed om six indi idual women/g oup a e shown.
Signi ican di e ences a e indica ed as ollows: *p < 0.05, ***p < 0.001 au ologous IP e sus
heal hy p egnan IP; ###p < 0.001 au ologous NIP e sus NIP om no mal p egnan women.

1
Table 1.
Clinical cha ac e is ics o non-p egnan (NP), heal hy p egnan (HP), and p eeclamp ic
p egnan (PE) women
Clinical da a
NP women
(n=35)
HP women
(n=31)
PE women
(n=39)
p alue
Age (yea s)
31.6  2.8
31.1  4.3
31.3  3.9
0.05
Ges a ional age a blood-
sampling (weeks)
NA
32.1  3.3
32.4  4.9
0.05
Ges a ional age a
deli e y (weeks)
NA
40.0  1.4
37.4  2.3
<0.05*
P e-p egnancy BMIb
(kg/m2)
NA
24.3  1.4
24.6  1.6
0.05
BMI a blood sampling
(kg/m2)
25.2  1.4
27.4  4.4
30.8  4.2
<0.05*
Pa i y a
NA
0 (0–2)
0 (0–1)
0.05
G a idi y a
NA
2 (1–3)
2 (1–3)
0.05
Sys olic blood p essu e a
blood sampling (mm Hg)
124  4.7
121  5.8
153  7.8
<0.05*
Dias olic blood p essu e
a blood sampling (mm
Hg)
80  4.6
79  6.5
104  4.1
<0.05*
Neona al weigh (g)
NA
3533.1  538
3008  736
<0.05*
P o einu ia a blood
Sampling, u ine dips ick a
0 (0–0)
0 (0–0)
3 (1–3+)
<0.05*
Mean alues  SD a e p esen ed. aValues a e exp essed as median ( ange). NA: no
applicable. Signi ican di e ences a e indica ed as ollows: *p<0.05 heal hy p egnan e sus
p eeclamp ic p egnan women, bBMI: body mass index
Table 1
Figu e 1.
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
au ologous plasma he e ologous
plasma heal hy p egnan
plasma p eeclamp ic
p egnan plasma
O2-. p oduc ion [nmol/min/3x105 cells]
IP
NIP
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
au ologous plasma he e ologous
plasma heal hy p egnan
plasma p eeclamp ic
p egnan plasma
O2-. p oduc ion [nmol/min/3x105 cells]
IP
NIP
***
###
A
##
B
Figu e 1
Figu e 2.
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
au ologous plasma he e ologous
plasma heal hy non-
p egnan plasma p eeclamp ic
p egnan plasma
O2-. p oduc ion [nmol/min/3x105 cells]
IP
NIP
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
au ologous plasma he e ologous
plasma heal hy non-
p egnan plasma p eeclamp ic
p egnan plasma
O2-. p oduc ion [nmol/min/3x105 cells]
IP
NIP
A
B
***
***
###
###
###
###
Figu e 2
1
Figu e 3.
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
au ologous plasma he e ologous
plasma heal hy non-
p egnan plasma heal hy p egnan
plasma
O2-. p oduc ion [nmol/min/3x105 cells]
IP
NIP
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
au ologous plasma he e ologous
plasma heal hy non-
p egnan plasma heal hy p egnan
plasma
O2-. p oduc ion [nmol/min/3x105 cells]
IP
NIP
A
B
###
*
###
Figu e 3