Comparative Analysis of the Maternal and Umbilical Interleukin-8 Levels in Normal Pregnancies and in Pregnancies Complicated By Preeclampsia With Intrauterine Normal Growth and Intrauterine Growth Retardation

By Laskowska, Marzena Laskowska, Katarzyna; Leszczynska-Gorzelak, Bozena; Oleszczuk, Jan

Abstract Objectives. The aim of this study was to determine the maternal and umbilical cord serum levels of interleukin-8 (IL-8) in pregnancies complicated by preeclampsia with intrauterine normal growth and intrauterine growth retardation (IUGR), and in normotensive pregnancies.

Patients and methods. The study was carried out on 15 patients with singleton pregnancies complicated by preeclampsia with appropriate for gestational age weight infants and 12 pregnant patients with preeclampsia complicated by ITJGR. The control group consisted of 10 healthy normotensive delivering patients with singleton uncomplicated pregnancies. Maternal and umbilical serum IL- 8 concentrations were estimated using the ELISA method.

Results. There were no statistically significant differences in patient profiles between the groups. Systolic and diastolic blood pressure and mean arterial blood pressure were higher in the study groups in comparison with the control group. Lower birth weight and lower gestational age at birth were observed in the group of patients with preeclampsia complicated by IUGR. Increased maternal and umbilical serum levels of IL-8 were found in both preeclamptic patient groups in comparison with the control group. The umbilical cord blood concentrations of IL-8 in all groups of patients tended to be higher in comparison with the maternal blood.

Conclusions. It seems that these higher IL-8 concentrations may be associated with apoptosis, inflammation, neutrophil activation, endothelial cell damage and dysfunction, and increased endothelial permeability. They may also participate in an attempt to compensate for the imbalanced apoptosis and vascular resistance. Our findings suggest a possible significant role of IL-8 in the pathogenesis and sequelae of preeclampsia, especially in preeclamptic pregnancies complicated by IUGR.

Keywords: Interleukin-8, preeclampsia, IUGR, maternal and umbilical blood

Introduction

In preeclamptic pregnant women, the invasion of trophoblast and physiologic remodeling of the spiral arteries, characteristic of normal pregnancy, are inadequate. It seems that disorders in the vascular endothelium and in placental and uteroplacental vascular system development may be the primary causes of preeclampsia.

Incubation of myometrial vessels obtained from women with uncomplicated pregnancies with plasma from preeclamptic women significantly reduced endothelium-dependent relaxation compared with vessels incubated with plasma from normal pregnant women [1]. These observations support the hypothesis that altered concentrations of various cytokines might explain the shallow placentation and endodielial cell dysfunction [1,2] that play a crucial role in the pathogenesis of preeclampsia, possibly reflecting a condition of persistent inflammation. Inflammatory reactions and neutrophil and monocyte activation have also been observed in pregnancies complicated by preeclampsia or intrauterine growth retardation (IUGR) [3]. Preeclampsia is also characterized by neutrophil and monocyte activation and enhanced chemokine activation [4,5]. Halim et al. observed a cytokine- and neutrophil-mediated liver injury accompanying the HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count) [6].

Interleukin-8 (IL-8) is a strong neutrophil chemoattractant and activator, and it is necessary for monocyte recruitment to the vascular endothelium [7]. IL-8/neutrophil-activating peptide- 1 (NAP- I) selectively stimulates the ability of neutrophils and T- lymphocytes to invade injured or inflamed tissue [8,9] . Study findings indicate that IL-8 may participate in the pathogenesis of adult respiratory distress syndrome, bacterial infections, graft rejection, glomerulonephritis, and placental infection [10,11]. The property of IL-8 to stimulate movement of neutrophils across endothelial monolayers in vitro supports the concept of a central role of this molecule in the accumulation of neutrophils in inflammatory lesions in vivo.

Besides its chemotactic effect, IL-8 displays other distinct characteristics. In neutrophils it triggers the secretion of superoxide anions and lysosomal enzymes, thereby indirectly augmenting the permeability of blood vessels [12]. Other findings suggest that endothelium-derived IL-8 may function to attenuate the inflammatory effect at the interface between vessel wall and blood, by inhibiting neutrophil adhesion to cytokine-activated endothelial monolayers. Therefore these cells seem to be protected against neutrophil-mediated damage [13].

Endothelial cell expression of IL-8 mRNA was enhanced in response to the incubation with fetal plasma from placental vascular disease in comparison to incubation with fetal plasma from normal pregnancy [14]. Furthermore, IL- 8 levels correlated positively with both TNF- alpha and von Willebrand factor. Higher levels of IL-8 may result from the increased production of IL-8, which is secondary to the increased TNF-alpha level and/or reduced clearance [4].

Uterine chemokines, apart from the regulation of leukocyte migration and function, also display specific roles in endometrial angiogenesis, apoptosis, proliferation, and differentiation. Coordination of chemokine-chemokine receptor interactions plays an important role in the menstrual cycle and successful pregnancy. Moreover, unbalanced chemokine expression contributes to pathological conditions characterized by uncontrolled cellular proliferation, migration and invasion [15].

The aim of this study was to carry out a comparative analysis of the maternal and umbilical cord serum levels of IL-8 in pregnancies complicated by preeclampsia with normal intrauterine fetal growth, in preeclamptic pregnancies with IUGR, and in normo tensive pregnancies.

Patients and methods

Fifteen patients with singleton pregnancies complicated by preeclampsia with appropriate for gestational age weight infants (group P) and 12 pregnant patients with preeclampsia complicated by IUGR (group PI) were included in the study. Preeclampsia was defined as blood pressure of at least 140/90 mmHg and proteinuria of at least 2+ on dipstick testing, each on two occasions 4-6 hours apart. Severe preeclampsia was defined as blood pressure > 160/110 mmHg on at least two occasions 6 hours apart, proteinuria >5 g in a 24-hour urinary protein excretion, or HET ,T P syndrome. A birth weight below the 10th percentile for gestational age was classified as IUGR.

The control group consisted of 10 healthy normotensive delivering patients with singleton uncomplicated pregnancies, without any renal, heart or vascular diseases and with normal laboratory tests (group C). All arterial blood pressure values in the control group were normal and did not exceed 135/ 85 mmHg. None of the patients from this group suffered from proteinuria. All patients in the study were non-smokers.

Five milliliters of blood were taken by venipuncture from each preeclamptic patient and from each woman in the control group before the active phase of labor, and collected in sterile tubes. Five milliliters of umbilical vein blood were taken immediately after delivery and collected in sterile tubes. They were centrifuged for 15 min at 500 x g after sampling. Each serum obtained was frozen at – 70[degrees]C until assayed.

Maternal and umbilical serum IL-8 concentrations were estimated using a sandwich ELISA assay according to the manufacturer’s instructions (human IL-8/NAP-1 ELISA kit, Bender MedSystems GmbH, Vienna, Austria).

The study was given the approval of the board for supervising ethics in medical experiments at the university medical school.

Data are expressed as mean + standard deviation. Statistical analysis was undertaken using the computer program Statistica 5.0 using the Shapiro-Wilk test for normal distribution of data, the Levene test for equality of variance, and subsequendy Student’s r- tests or (in unequal variance) the Cochran-Cox test, the Mann- Whitney U test (absence of normal distribution and non-parametric data), and the Rruskal-Wallis test in independent samples, and the i- test or Wilcoxon rank sum test for data-dependent samples in one group of patients. The level of statistical significance was established as p < 0.05.

Results

There were no statistically significant differences in patient profiles between groups in gravidity, parity or maternal age. Creatinine and urea levels were normal in all patients. None of the patients from the control group suffered from proteinuria. There were no statistically significant differences in patient profiles between groups in mode of delivery. The mode of labor had no effect on umbilical cord serum levels of IL-8 in any of the studied groups of patients. Systolic and diastolic blood pressure and mean arterial blood pressure were higher in the study groups in comparison with the control group. These differences were statistically significant (p < 0.001) (Table I).

The mean systolic blood pressure values were 154.38 +-18.41 mmHg in group P, 158.83 +- 21.37 mmHg in group PI, and 105.91 +- 10.68 mmHg in the control group C. The mean diastolic blood pressure values were 106.88 +- 12.23 mmHg in group P, 105.83 +- 12.42 mmHg in group PI, and 67.73 +- 8.17 mmHg in the controls. These differences were statistically significant (p < 0.001). Lower birth weight and lower gestational age at birth were observed in the group of patients with preeclampsia complicated by IUGR in comparison with the two other groups of patients studied (preeclamptic patients with normal intrauterine fetal growth and the control group; Table I).

Increased maternal serum levels of IL-8 were found in both groups of preeclamptic patients in comparison with the control group. The mean maternal values of IL-8 were 79.00 +- 150.10 pg/ mL in preeclamptic patients with normal fetal growth (group P), 107.02 +- 221.75 pg/mL in preeclamptic patients with IUGR (group PI), and 43.00 +- 35.63 pg/mL in healthy controls. These differences were not statistically significant (Figure 1).

Table I. Analysis of obtained results.

Figure 1. Interleukin-8 levels in maternal blood.

There were higher levels of IL-8 in the umbilical serum in preeclamptic patients with normal fetal growth than in the controls, but these differences were not statistically significant. The mean value in umbilical blood in preeclamptic patients with normal fetal growth (group P) was 190.78 +- 326.04 pg/mL.

There were higher levels of IL-8 in the umbilical cord blood in preeclamptic patients with IUGR than in the control group. These differences were statistically significant (p = 0.04). The mean values of umbilical serum levels of IL-8 were 288.79 +- 372.11 pg/ mL in group PI and 126.44 +- 249.87 pg/ mL in the controls (Figure 2).

There were higher levels of IL-8 in the maternal and umbilical cord blood in preeclamptic patients with retarded fetal growth (group PI) than in preeclamptic patients with normal fetal growth (group P). But these differences were not statistically significant (p = 0.28 and ? = 0.24, respectively). The umbilical cord blood concentrations of IL-8 in all groups of patients tended to be higher in comparison with the maternal blood.

Discussion

Preeclampsia is characterized by an increase in vascular tone, hypertension, enhanced platelet aggregation and coagulation, and decreased intravascular volume due to increased endothelial permeability [16]. All these abnormalities can be attributed to endothelial cell damage and dysfunction and may be associated with abnormal production of cytokines.

Disturbances in cytokine production may be the cause of the pathological structure and function of the placenta and may result in the pathology of pregnancy, such as preeclampsia and IUGR.

IL-8 may play a crucial role in the regulation and the course of normal and pathological pregnancies. In our study a higher increase in IL-8 concentrations was observed in preeclampsia complicated with IUGR than in preeclampsia with normal fetal growth. These results may suggest differences in die time of onset (earlier) and the enhancing of these disturbances in preeclampsia with IUGR compared with preeclampsia with normal fetal growth.

Figure 2. Interleukin-8 levels in umbilical cord blood.

Similar results were presented by Zhang et al. [17], who observed higher concentrations of IL-8 in blood from preeclamptic patients than in samples from non-pregnant women and from healthy pregnant patients. These researchers observed increased endothelial cell monolayer permeability after inducing with preeclamptic serum. According to the authors it appears that increased lipid peroxides, which alter barrier function of the endodielial cell monolayer in preeclampsia, may be associated with endothelial cell oxidative stress and increased levels of IL-8, which may be a factor altering endothelial cell barrier function.

Also Johnson et al. [18] observed elevated levels of IL-8 in pregnancies complicated by preeclampsia, but in normotensive pregnancies complicated by IUGR the levels were normal. These findings suggest that the observed changes result from hypertension and preeclampsia [18]. Our findings suggest that these changes are probably more enhanced in preeclamptic pregnancy complicated by IUGR.

Contrasting results were obtained by Greer et al. [19], who reported no significant changes in concentrations of IL-8 in preeclamptic patients compared to normal patients.

Endothelial cells are considered a key link between reduced perfusion and injury of maternal organs, and placental disturbances observed in preeclampsia. Vascular endothelial cells play a very important role in normal placental function and normal reactivity and sensitivity of adjacent vascular smooth muscle.

Buemi et al. [20] observed that IL-8 and adhesion molecule ICAM- I significantly enhanced the Ca^sup 2+^dependent K+ outflow in red blood cells from hypertensive subjects, but had an inhibitory effect on cells from controls. Vasoconstriction in subjects witii essential hypertension may therefore depend on different regulation of ionic flow that probably supports an increased Ca^sup 2+^ inflow in smooth muscle fiber cells. Under certain pathological conditions, some immune system components (i.e., interleukins, adhesion molecules) may directly enhance membrane permeability to Ca^sup 2+^, thus inducing vasoconstriction in the smooth muscle cells. The imbalance between vasodilators and vasoconstrictors observed in pregnancy complicated by preeclampsia may result in IUGR. Also IL-8 in this pathological condition in preeclampsia may directly enhance membrane permeability to Ca^sup 2+^, thus inducing vasoconstriction in the smooth muscle cells.

Wang et al. [21] observed significantly decreased placental production of IL-8 and 6-ketoprostaglandin F1alpha (6-keto- PGF1alpha) in preeclampsia compared with normal pregnancies. This placental production of 6-keto-PGF1alpha and IL-8 was significantly correlated in preeclampsia. Furthermore placental tissue treated with IL-8 exhibited a concentration-dependent increase in 6-keto- PGF1alpha production.

If IL-8 improves placental prostacyclin production in preeclampsia, our findings about the increased IL-8 concentrations may suggest die presence of a compensation mechanism or an attempt to compensate for existing disorders in pregnancy complicated by preeclampsia. Enhanced production of IL-8 seems to suggest also the attempt to stimulate prostacyclin production in order to compensate for its decreased production and to restore the normal balance between vasodilators and vasoconstrictors.

Gimbrone et al. [13] suggest that endothelial-derived IL-8 may function to attenuate inflammatory events at the interface between vessel wall and blood by inhibiting neutrophil adhesion to cytokine- activated endothelial monolayers, and it may protect these cells against neutrophil-mediated damage [13]. The inflammatory response is a major component in the patiiophysiology of preeclampsia and IUGR [22]. Endothelium is a main connection between placental ischemia and clinical manifestation during vascular pregnancy complications [22] .

Abdollahi et al. [23] observed that IL-8 blocks tumor necrosis factor-related apoptosis-inducing Iigand (TRAIL) -induced cell death in an ovarian carcinoma cell line and was able to turn the TRAIL- sensitive cell into a TRAIL-resistant one. They have also shown that TRAIL was able to induce caspase-8 cleavage in diese cells, whereas pretreatment with IL-8 blocked this caspase cleavage. Our results and other reports suggest that the imbalance in proliferation and apoptosis in preeclampsia with and without IUGR may be associated with the alteration of IL-8 levels. It seems that these higher IL-8 concentrations might also be associated with inflammation, neutrophil activation, endothelial cell damage and dysfunction, increased endothelial permeability, and vascular resistance. Furthermore higher levels of IL-8 might also be considered as protective and compensative mechanisms against disorders observed in pregnancy complicated by preeclampsia with and without IUGR.

It is possible that higher levels of maternal and umbilical IL-8 in preeclampsia with appropriate intrauterine fetal growth and additionally higher levels of IL-8 in pregnancies complicated by IUGR in the course of preeclampsia may be considered as the stage of progress of the same disease or another time of onset of this disease. Our observations may suggest more advanced pathological changes in the placental and fetal circulatory system in pregnancies complicated by IUGR in the course of preeclampsia, resulting in inadequate fetal oxygenation and nutrition, and consequently leading to fetal hypotrophia in these pregnancy disorders. Further studies are necessary to evaluate the role of increased IL-8 levels in preeclamptic pregnancies with normal and growth-retarded fetuses.

Acknowledgements

This work was supported by research grant from Polish State Committee for Scientific Research (KBN).

References

1 . Myers J, Mires G, Macleod M, Baker P. In preeclampsia, the circulating factors capable of altering in vitro endothelial function precede clinical disease. Hypertension 2005;45:258-263.

2. Madazali R, Aydin S, Uludag S, Vildan O, Tolun N. Maternal plasma levels of cytokines in normal and preeclamptic pregnancies and their relationship with diastolic blood pressure and fibronectin levels. Acta Obstet Gynecol Scand 2003;82:797-802.

3. Redman CW, Sacks GP, Sargent IL. Preeclampsia: An excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol 1999;180:499-506.

4. Velzing-Aarts FV, Muskiet FA, van der Dijs FP, Duits A. High serum interleukin-8 levels in Afro-Caribbean women with pre- eclampsia. Relations with tumor necrosis factor-alpha, Duffy negative phenotype and von Willebrand factor. Am J Reprod Immunol 2002;48:319-322.

5. Mellembakken JR, Aukrust P, Hestdal K, Ueland T, Abyholm T, Videm V. Chemokines and leukocyte activation in the fetal circulation during preeclampsia. Hypertension 2001;38:394-398.

6. Halim A, Kanayama N, Maradny E, Maehara K, Takahashi A, Nosaka K, Fukuo S, Amamiya A, Kobayashi T, Terao T. Immunohistological study in cases of HET ,1 ,P syndrome (hemolysis, elevated liver enzymes and low platelets) and acute fatty liver of pregnancy. Gynecol Obstet Invest 1996;41:106-112. 7. Kauma S, Takacs P, Scordalakes C, Walsh S, Green K, Peng T. Increased endothelial monocyte chemoattractant protein-1 and interleukin-8 in preeclampsia. Obstet Gynecol 2002;100:706-714.

8. Leonard EJ. NAP-I QL-S). Immunol Today 1990;11:223224.

9. Smith WB, Gamble JR, Clark-Lewis I, Vadas MA. Interleukin-8 induces neutrophil transendothelial migration. Immunology 1991;72:65- 72.

10. Puchner T, Egarter C, Wimmer C, Lederhilger F, Weichselbraun I. Amniotic fluid interleukin-8 as a marker for intraamniotic infection. Arch Gynecol Obstet 1993;253: 9-14.

11. Tilg H, Ceska M, Vogel W, Herold M, Margreiter R, Huber C. Interleukin-8 serum concentrations after liver transplantation. Transplantation 1992;53:800-803.

12. Sticherling M, Bornscheuer E, Schroder JM, Christophers E. Localization of neutrophil-activating peptide-l/interleukin-8 immunoreactivity in normal and psoriatic skin. J Invest Dermatol 1991;96:26-30.

13. Gimbrone MA, Obin MS, Brock AF, Luis EA, Haas PE, Hebert CA, Yip YK, Leung DW, Lowe DG, Kohr WJ, et al. Endothelial interleukin- 8: A novel inhibitor of leukocyte-endothelial interactions. Science 1989;246:1601-1603.

14. Wang X, Athayde N, Trudinger B. Fetal plasma stimulates endothelial cell production of cytokines and the family of suppressor of cytokine signaling in umbilical placental vascular disease. Am J Obstet Gynecol 2003;188:510-516.

15. Kayisli UA, Mahutte NG, Arici A. Uterine chemokines in reproductive physiology and pathology. Am J Reprod Immunol 2002;47:213-221.

16. Wang Y, Gu Y, Zhang Y, Lewis DF. Evidence of endothelial dysfunction in preeclampsia: Decreased endothelial nitric oxide synthase expression is associated with increased cell permeability in endothelial cells from preeclampsia. Am J Obstet Gynecol 2004;190:817-824.

17. Zhang Y, Gu Y, Li H, Lucas MJ, Wang Y. Increased endothelial monolayer permeability is induced by serum from women with preeclampsia but not by serum from women with normal pregnancy or that are not pregnant. Hypertens Pregnancy 2003;22:99-108.

18. Johnson MR, Anim-Nyame N, Johnson P, Sooranna SR, Steer PJ. Does endothelial cell activation occur with intrauterine growth restriction? BJOG 2002;109:836-839.

19. Greer IA, Lyall F, Perera T, Boswell F, Macara LM. Increased concentrations of cytokines interleukin-6 and interleukin-1 receptor antagonist in plasma of women with preeclampsia: A mechanism for endothelial dysfunction? Obstet Gynecol 1994;84:937-940.

20. Buemi M, Marino D, Floccari F, Ruello A, Nostro L, Aloisi C, Marino MT, Di Pasquale G, Corica F, Frisine N. Effect of interleukin- 8 and ICAM-I on calcium-dependent outflow of K+ in erythrocytes from subjects with essential hypertension. Curr Med Res Opin 2004;20:19- 24.

21. Wang X, Baier J, Adair CD, Lewis DF, Krueger S, Kruger T, Gurski M, Brown E. Interleukin-8 stimulates placental prostacyclin production in preeclampsia. Am J Reprod Immunol 1999;42:375-380.

22. Bretelle F, Sabatier F, Shojai R, Agostini A, Dignat-George F, Blanc B, d’Ercole C. [New insight in physiopathology of preeclampsia and intra-uterine growth retardation: Role of inflammation] [Article in French]. Gynecol Obstet Fertil 2004;32:482- 489.

23. AbdoUahi T, Robertson NM, Abdollahi A, Litwack G. Identification of interleukin-8 as an inhibitor of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in ovarian carcinoma cell line OVCAR3. Cancer Res 2003;63:4521-4526.

MARZENA LASKOWSKA1, KATARZYNA LASKOWSKA2, BOZENA LESZCZYNSKA- GORZELAK1, & JAN OLESZCZUK1

1 Department of Obstetrics and Perinatology, University School of Medicine in Lublin, Poland, and 2 Department of

Gastroenterology, University School of Medicine in Lublin, Poland

(Received 24 October 2006; revised 29 January 2007; accepted 7 March 2007)

Correspondence: Marzena Laskowska, MD, PhD, Chair and Department of Obstetrics and Perinatology, University School of Medicine in Lublin, Jaczewskiego 8, 20-950 Lublin, Poland. Tel/Fax: +48 81 7425235. E-mail: melaskowska@go2.pl

Copyright Taylor & Francis Ltd. Jul 2007

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