Granulocyte superoxide anion production and regulation by plasma factors in normal and preeclamptic pregnancy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 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. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 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; 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6 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., 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 8 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 9 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 16 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. 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Neu ophils, molecules, unc ions and pa hophysiological aspec s. Lab. In es . 80, 617-653. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 20 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. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 21 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