- Email: [email protected]
Meconium-stained amniotic fluid exhibits chemotactic activity for polymorphonuclear leukocytes in vitro
Journal of Reproductive Immunology 46 (2000) 21 – 30 www.elsevier.com/locate/jreprimm
Meconium-stained amniotic fluid exhibits chemotactic activity for polymorphonuclear leukocytes in vitro Tetsuo Yamada, Hisanori Minakami *, Shigeki Matsubara, Tomomi Yatsuda, Yasuhiro Kohmura, Ikuo Sato Center for Perinatal Medicine, Jichi Medical School Hospital, Minamikawachi-machi, Tochigi, 329 -0498 Japan Received 18 May 1999; received in revised form 18 July 1999; accepted 12 August 1999
Abstract We have studied whether meconium-stained, turbid amniotic fluid (turbid AF) obtained during term pregnancy possesses chemotactic activity for polymorphonuclear leukocytes (PMNs) in the absence of clinically apparent infection. Eight samples of turbid AF were obtained from eight women who underwent a cesarean section (four emergency and four elective cesarean sections) in the absence of signs of clinical infection or fetal distress. Samples of clear AF obtained from nine women during an elective cesarean section served as a control. We used also a negative control (medium only) and a positive control containing 10 nM N-formyl-methionyl-leucyl-phenylalanine. The control or turbid AF specimen was placed in the lower compartment of a blind well chamber, and the PMN suspension was placed in the upper compartment. Following incubation, the number of PMNs that had migrated and passed through the filter to the AF was counted. The number of control PMNs that migrated to the turbid AF (2009 59) was comparable to that of the positive (162924) but significantly exceeded that of the clear AF (17 9 11; PB 0.0001) and of the negative control (25 99; PB 0.0001). Checkerboard assay indicated that the turbid AF exhibited a dose-dependent chemotactic activity for PMNs. The turbid AF contained higher levels of TNFa, IL-1b and IL-8 than the clear AF. The concentration of IL-8 in the AF was correlated positively with the chemotactic activity of the AF (r =0.733, P= 0.0005). Anti-human IL-8 antibody added in the turbid AF dose-dependently abolished the chemotactic
* Corresponding author. Tel.: + 81-285-442111; fax: + 81-285-448505. E-mail address: [email protected] (H. Minakami) 0165-0378/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 0 3 7 8 ( 9 9 ) 0 0 0 4 8 - 0
22
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
activity of the turbid AF. It is concluded that meconium-stained AF is a chemoattractant for PMNs and that cytokines such as an IL-8 may be involved in this phenomenon. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Amniotic fluid; Chemotaxis; Leukocyte; Meconium; Term labour
1. Introduction Staining of the amniotic fluid (AF) with meconium (turbid AF) reportedly occurs in 7–25% of term pregnancies (Katz and Bowes, 1992; Berkus et al., 1994). The adverse effects of meconium on the infant include pulmonary disease, pulmonary hypertension of the newborn, and pneumothorax (Katz and Bowes, 1992; Nathan et al., 1994), while the mother is at an increased risk of postpartum infection (Markovitch et al., 1993; Piper et al., 1998). Reports of any possible beneficial effect of meconium on the infant are limited (Montgomery et al., 1995). In an in vitro study, Montgomery et al. (1995) observed that meconium inhibited the contractile effect of the thromboxane A2 analogue U46619, in the umbilical artery and in venous smooth muscle, which suggests that meconium can neutralize certain vasoconstrictor stimuli in utero. The passage of meconium may result from fetal asphyxia in some patients. If the fetal demand for oxygen exceeds the placental ability to supply it, the fetus is at an increased risk for hypoxia and it may defecate meconium into the AF. In the presence of insufficient placental function, the spontaneous onset of the labour may result in the live birth of the infant. Term parturition is associated with an increased concentration of neutrophil attractant in the AF (Romero et al., 1991). Our hypothesis is that the turbid AF possesses a stronger chemotactic activity for polymorphonuclear leukocytes (PMNs) than the clear AF in term pregnancy.
2. Material and methods
2.1. Study subjects and preparation of amniotic fluid (AF) After obtaining informed consent, specimens of AF were collected during cesarean section performed in 17 women for an obstetrical indication other than fetal distress. Samples were obtained from nine women who underwent an elective cesarean section at 38.091.2 weeks of gestation. Turbid AF were obtained from eight women: four underwent an elective cesarean section at 39.09 1.8 weeks of gestation and had light meconium; four underwent an intrapartum cesarean section for dystocia at 39.090.8 weeks
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
23
of gestation and had thick meconium. In all cases, the maternal serum level of C-reactive protein on the day before the cesarean section was B0.4 mg/dL. On the day of cesarean section, each woman exhibited a normal body temperature. Twelve women who underwent an elective cesarean section had no labour pains. The 17 neonates born to these mothers had no signs of infection and did well during their hospital stay. The mean birthweight of the nine infants with clear AF and of the eight infants with turbid AF did not differ significantly: 29259289 g vs. 30359450 g, respectively. Part of each specimen of AF was cultured for aerobic and anaerobic bacteria, Mycoplasma hominis, Ureaplasma urealyticum, Candida species, and Chlamydia trachomatis. The remainder of each sample was centrifuged at 1500× g for 10 min and the supernatant was used in studies of clear or turbid AF. As the negative control, we used RPMI-1640 (Gibco BRL, USA) and, as the positive control, RPMI-1640 containing 10 nM N-formylmethionyl-leucyl-phenylalanine (fMLP, Peninsula Laboratories, Belmont, USA), which is a known chemoattractant for PMNs (Williams et al., 1977). Samples and controls were stored at −80°C until assayed. Four groups of specimens were prepared: clear AF (n=9), turbid AF (n=8), negative control (n=6), and positive control (n= 6). PMNs were obtained from three healthy volunteer women with uncomplicated singleton pregnancies at term. PMNs were isolated according to the method of Dooley et al. (1982), and were suspended at a concentration of 5×106/ml in RPMI-1640 with 10% fetal calf serum (FCS).
2.2. Chemotactic assay and checkerboard assay The in vitro chemotactic activities of the clear AF in the presence or absence of exogenous IL-8 (recombinant human IL-8; R& D Systems, Minneapolis, USA), the turbid AF in the presence or absence of monoclonal anti-human IL-8 antibody (R& D Systems, Minneapolis, USA), the negative control, and the positive control for PMNs were measured using a modified blind well chamber (Neuro Probe, Washington, DC) employing Boyden’s technique, as described by Ward et al. (1965). The chamber had two compartments separated by a polycarbonate filter, having a pore diameter of 3 mm. An aliquot of 200 ml of the AF or control was placed in the lower compartment, and a 100 ml suspension of PMNs was placed in the upper compartment. After incubation for 40 min at 37°C in 5% CO2 in air, the PMNs that migrated to the lower side of the filter were stained with Diff-Quik (Kokusaisiyaku, Kobe, Japan) and their number in five randomly selected high-power fields (× 400) was counted under a light-microscope. Chemotactic activity was expressed as the number of PMNs counted. All
24
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
assays were performed in duplicate. To confirm the chemotactic activity of turbid AF for PMNs, a checkerboard assay was performed according to the method of Zigmond and Hirsch (1973). Various concentrations of the turbid AF (0–64% in final) were added to the PMN suspension in the upper compartment and to the negative control (RPMI-1640 with 10% FCS) in the lower compartment.
2.3. Assay of TNFa, IL-1b, and IL-8 in AF The concentrations of TNFa, IL-1b, and IL-8 were measured in AF using commercially available kits (Human TNFa Immunoassay, R& D systems, Minneapolis, USA; Human IL-1b (J Immunoassay, R& D systems, Minneapolis, USA; and Human interleukin-8 ELISA kit, Toray-Fuji Bionics, Tokyo, respectively) according to the manufacturer’s instruction. The minimum detectable concentration for TNFa, IL-1b and IL-8 was 0.2, 0.1, and 1.1 ng/l, respectively. The intra- and inter-assay coefficients of variation for each cytokine were B9 and 12%, respectively.
2.4. Statistical analysis Data are reported as mean91 SD. Wilcoxon rank-sum test was used to compare means. Spearman rank correlation coefficients were determined to analyze the linear regression between the number of PMNs migrated and the concentration of TNFa, IL-1b, or IL-8. A level of PB0.05 was accepted as statistically significant.
3. Results Results of bacterial culture were negative in all 17 samples of AF. The Diff-Quik-stained PMNs that migrated from the upper compartment and were trapped on the filter of the lower compartment side are illustrated in Fig. 1. The number of PMNs (200959) that migrated from the upper compartment in which suspension of PMNs was placed to the turbid AF in the lower compartment was comparable to that of the positive control (1629 24), but was significantly higher than that of the clear AF (17911, P B 0.0001) as well as that of the negative control (2599, PB0.0001) (Fig. 2). Although four samples of the eight turbid AF were obtained from women without labour pains, the chemotactic activity for PMNs of these AF samples was comparable with that of the AFs obtained from four women during an intrapartum cesarean section.
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
25
To test the chemotactic activity of turbid AF for PMNs, a checkerboard assay was performed (Fig. 3). In the presence of 64% turbid AF (final concentration) in the lower compartment, 200, 175, 108, and 39 PMNs migrated from the upper compartment in which 0, 4, 16, and 64% turbid AF was present, respectively, to the lower compartment. In the absence of turbid AF in the lower compartment, varying concentrations of turbid AF in the upper compartment did not influence the number of migrated PMNs and only 20–39 PMNs migrated. In the absence of turbid AF in the upper compartment, the number of migrated PMNs increased with increasing concentration of turbid AF in the lower compartment. Thus, the results of the checkerboard assay indicated that the concentration gradient of turbid AF was important in the migration of PMNs, which confirmed that turbid AF possessed a dose-dependent chemotactic activity for PMNs. With a negative gradient of turbid AF, in which the upper compartment contained less turbid AF than the lower compartment, the migration of PMNs from the upper to the lower compartment increased in accordance with an increase in the difference in concentration of turbid AF between the two chambers. In contrast, a positive gradient of turbid AF between the two chambers did not enhance the migration of PMNs.
Fig. 1. Photomicrographs showing polymorphonuclear leukocytes (PMNs) that had migrated to: (a) the negative control; (b) the positive control; (c) clear amniotic fluid; and (d) meconium-stained (turbid) amniotic fluid. Arrows in (a, d) indicate leukocytes. Two leukocytes are observed in (a), while about 50 are present in (d). Filter pores are indicated by arrowheads.
26
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
Fig. 2. Diagram showing the number of polymorhonuclear leukocytes (PMNs) that had migrated to the negative (n= 6) and positive controls (n= 6), clear amniotic fluid (n= 9) and meconium-stained (turbid) amniotic fluid (n= 8). Closed triangles indicate the negative and positive controls. Open circles indicate the AF obtained from women who underwent an elective cesarean section in the absence of labour pains. Closed circles indicate the AF obtained from women who underwent an intrapartum cesarean section.
Concentrations of TNFa, IL-1b and IL-8 were measured in nine samples of clear AF and eight samples of turbid AF. The turbid AF contained higher levels of TNFa, IL-1b and IL-8 than did the clear AF (P B 0.0001) (Fig. 4). No overlap in the concentration of any of these three cytokines was observed between the clear and the turbid AF. Turbid AF obtained during an intrapartum cesarean section tended to contain a higher level of each cytokine than did turbid AF obtained during an elective cesarean section. A strongest significant correlation was observed between the number of PMNs migrated and the concentration of IL-8 in the AF. A significant correlation was observed also between the concentrations of IL-1b and IL-8 in the AF (data not shown; r=0.936, P B 0.0001). To examine whether IL-8 was responsible for the chemotactic activity of turbid AF, varied concentrations of recombinant IL-8 (rIL-8) were added to the clear AF or varied concentrations of anti-human IL-8 antibody were added to the turbid AF. The chemotactic activities of these modified AFs were then examined. Addition of rIL-8 to the clear AF conferred dose-dependent chemotactic activity for PMNs on the clear AF (Fig. 5A). Addition of anti-human IL-8 antibody in the turbid AF dosedependently abolished the chemotactic activity of the turbid AF (Fig. 5B).
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
27
4. Discussion The present study has demonstrated that meconium-stained, turbid, AF exhibits a biological neutrophil attractant activity equal to that of 10 nM fMLP in the absence of bacteria and clinical infection. In such AF that possessed neutrophil attractant activity, levels of the so-called inflammatory cytokines such as TNFa, IL-1b, and IL-8 were markedly increased. Among cytokines examined in the present study, concentration of IL-8 in the AF had the strongest correlation with the chemotactic activity of the AF. IL-8 is released by many uterine tissues, including the placenta (Shimoya et al., 1992), the chorion-decidua (Kelly et al., 1992) and the cervix (Bardlay et al., 1993), and is a potent neutrophil attractant and activator (Baggiolini et al., 1989). IL-8 is reportedly produced by normal colonic epithelial cells (Patel et al., 1995) and was recently reported to be present in meconium, and has
Fig. 3. Effect of differing concentrations of turbid AF between the upper and lower compartments on the number of PMNs that migrated from the upper compartment to the lower compartment. Values represent the mean of duplicate experiments. In the presence of a higher concentration of the turbid AF in the lower compartment than in the upper compartment, a larger number of PMNs migrated from the upper compartment to the lower compartment.
28
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
Fig. 4. Relationships between the number of PMNs migrated and the concentration of TNFa, IL-1b or IL-8 in the AF. Clear AF () (n= 9), turbid AF obtained during an elective cesarean section in the absence of labour pains ( ) (n= 4), and turbid AF obtained during an intrapartum cesarean section ( ) (n= 4). A significant correlation was observed between the number of PMNs migrated and the concentration of IL-1b (r= 0.688, P = 0.0016), between the number of PMNs migrated and the concentration of IL-8 (r= 0.733, P = 0.0005), but not between the number of PMNs migrated and the concentration of TNFa (r=0.471, P =0.055).
been suggested to be responsible for the neutrophil attractant activity of meconium-stained AF (Beaufort et al., 1998). The results of these previous studies and our present findings suggest that turbid AF possesses a neu-
Fig. 5. Effects of exogenous recombinant IL-8 and anti-human IL-8 antibody on the chemotactic activity of AF. (A) Varied concentrations of IL-8 were added to the clear AF before chemotactic activity of the clear AF was assayed. (B) Varied concentrations of anti-human IL-8 antibody were added to the turbid AF before chemotactic activity of the turbid AF was assayed. Vertical bar indicates the mean 9 SD of triplicate experiments.
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
29
trophil attractant activity due to the action of the IL-8, although the exact source of IL-8 is not known. We did not examine histologically the placentas, chorion or uterine cervix of the 17 women studied. However, it has been reported that the presence of neutrophil attractant in AF is a more sensitive marker for histologicallyverified chorioamnionitis and early delivery than is the culture of AF (Cherouny et al., 1993). It is possible that a larger number of PMNs may have invaded these tissues of the women with turbid AF compared to those with clear AF. The PMNs that accumulate around the uterine cervix may play a role in ripening by causing collagenolysis (Junqueira et al., 1980; Rajabi et al., 1988; Osmers et al., 1992). The degree of neutrophil invasion of the cervical connective tissue reportedly parallels the clinical progression of cervical dilatation (Junqueira et al., 1980; Rajabi et al., 1988; Osmers et al., 1992). These findings suggest that the appearance of neutrophil attractant in the AF precedes the onset of frequent uterine contractions and labour pains, even in term labour, via the accumulation of PMNs in the absence of infection. It is not known whether such an accumulation of PMNs is required for the commencement of labour. In conclusion, although the exact source of IL-8 found in turbid AF is not determined, the meconium which contains IL-8 may exhibit neutrophil attractant activity in vivo once it is discharged into the AF. This phenomenon could be related indirectly to the commencement of labour pains.
Acknowledgements The authors are grateful to the Japanese Ministry of Health and Welfare for financial support (Pediatric Research Grant c 10-02).
References Baggiolini, M., Walz, A., Kunkel, S.L., 1989. Nerutrophil-activating peptide-1/interleukin-8, a novel cytokine that activates neutrophils. J. Clin. Invest. 84, 1045 – 1049. Bardlay, C.G., Brennand, J.E., Kelly, R.W., Galder, A.A., 1993. Interleukin-8 production by the human cervix. Am. J. Obstet. Gynecol. 169, 625 – 632. Beaufort, A.J., Pelikan, D.M.V., Elferink, J.G.R., Berger, H.M., 1998. Effect of interleukin 8 in meconium on in vitro neutrophil chemotaxis. Lancet 352, 102 – 105. Berkus, M.D., Langer, O., Samueloff, A., Xenakis, E.M.J., Field, N.T., Ridgeway, L.E, 1994. Meconium-stained amniotic fluid: increased risk for adverse neonatal outcome. Obstet. Gynecol. 84, 115–120.
30
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
Cherouny, P.H., Pankuch, G.A., Romero, R., Botti, J.J., Kuhn, D.C., Demers, L.M., Appelbaum, P.C., 1993. Neutrophil attractant/activating peptide-1/interleukin-8: association with histologic chorioamnionitis, preterm delivery, and bioactive amniotic fluid leukoattractants. Am. J. Obstet. Gynecol. 169, 1299 – 1303. Dooley, D.C., Simpson, J.F., Meryman, H.T., 1982. Isolation of large numbers of fully viable human neutrophils: a preparative technique using Percoll density gradient centrifugation. Exp. Hematol. 10, 591–599. Junqueira, L.C.U., Zugaib, M., Montes, G.S., Toledo, O.M.S., Krisztan, R.M., Shigihara, K.M., 1980. Morphological and histological evidence for the occurrence of collagenolysis and for the role of neutrophilic polymorphonuclear leukocytes during cervical ripening. Am. J. Obstet. Gynecol. 138, 273 – 281. Katz, V.L., Bowes, W.A., 1992. Meconium aspiration syndrome: reflections on a murky subject. Am. J. Obstet. Gynecol. 166, 171 – 183. Kelly, R.W., Leask, R., Galder, A.A., 1992. Choriodecidual production of interleukin-8 and mechanism of parturition. Lancet 339, 776 – 777. Markovitch, O., Mazor, M., Shoham-Vardi, I., Chaim, W., Leiberman, J.R., Glezerman, M., 1993. Meconium stained amniotic fluid is associated with maternal infectious morbidity in preterm delivery. Acta Obstet. Gynecol. Scand. 72, 538 – 542. Montgomery, L.D., Belfort, M.A., Saade, G.R., Moise, K.J. Jr., Vedernikov, Y.P., 1995. Meconium inhibits the contraction of umbilical vessels induced by thromboxane A2 analog U46619. Am. J. Obstet. Gynecol. 173, 1075 – 1078. Nathan, L., Leveno, K.J., Carmody, T.J., Kelly, M.A., Sherman, M.L., 1994. Meconium: a 1990s perspective on an old obstetric hazard. Obstet. Gynecol. 83, 329 – 332. Osmers, R., Rath, W., Adelmann-Grill, B.C., Fittkow, C., Kuloczik, M., Szeverenyi, M., 1992. Origin of cervical collagenase during parturition. Am. J. Obstet. Gynecol. 166, 1455–1460. Patel, R.T., Bain, I., Youngs, D., Keighley, M.R.B., 1995. Cytokine production in pouchitis is similar to that in ulcerative colitis. Dis Colon Rectum 38, 831 – 837. Piper, J.M., Newton, E.R., Berkus, M.D., Peairs, W.A., 1998. Meconium: a marker for peripartum infection. Obstet. Gynecol. 91, 741 – 745. Rajabi, M.R., Dean, D.D., Beydoun, S.M., Woessner, J.F., 1988. Elevated tissue levels of collagenase during dilation of uterine cervix in human parturition. Am. J. Obstet. Gynecol. 159, 971–976. Romero, R., Mazor, M., Avia, C., Ceska, M., Lindley, I., 1991. Neutrophil attractant/activating peptide-1/interleukin-8 in term and preterm parturition. Am. J. Obstet. Gynecol. 165, 813–820. Shimoya, K., Matsuzaki, N., Taniguchi, T., Kameda, T., Koyama, M., Neki, R., Saji, F., Tanizawa, O., 1992. Human placenta constitutively produces interleukin-8 during pregnancy and enhances its production in intrauterine infection. Biol. Reprod. 47, 220 – 226. Ward, P.A., Cochrane, C.G., Muller-Eberhard, H.J., 1965. The role of serum complement in chemotaxis of leukocytes in vitro. J. Exp. Med. 122, 327 – 346. Williams, L.T., Snyderman, R., Pike, M.C., Lefkowitz, R.J., 1977. Specific receptor sites for chemotactic peptides on human polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. USA 74, 1204–1208. Zigmond, S.H., Hirsch, J.G., 1973. Leukocyte locomotion and chemotaxis: new methods for evaluation and demonstration of a cell-derived chemotactic factor. J. Exp. Med. 137, 387–410.
.
Meconium-stained amniotic fluid exhibits chemotactic activity for polymorphonuclear leukocytes in vitro Tetsuo Yamada, Hisanori Minakami *, Shigeki Matsubara, Tomomi Yatsuda, Yasuhiro Kohmura, Ikuo Sato Center for Perinatal Medicine, Jichi Medical School Hospital, Minamikawachi-machi, Tochigi, 329 -0498 Japan Received 18 May 1999; received in revised form 18 July 1999; accepted 12 August 1999
Abstract We have studied whether meconium-stained, turbid amniotic fluid (turbid AF) obtained during term pregnancy possesses chemotactic activity for polymorphonuclear leukocytes (PMNs) in the absence of clinically apparent infection. Eight samples of turbid AF were obtained from eight women who underwent a cesarean section (four emergency and four elective cesarean sections) in the absence of signs of clinical infection or fetal distress. Samples of clear AF obtained from nine women during an elective cesarean section served as a control. We used also a negative control (medium only) and a positive control containing 10 nM N-formyl-methionyl-leucyl-phenylalanine. The control or turbid AF specimen was placed in the lower compartment of a blind well chamber, and the PMN suspension was placed in the upper compartment. Following incubation, the number of PMNs that had migrated and passed through the filter to the AF was counted. The number of control PMNs that migrated to the turbid AF (2009 59) was comparable to that of the positive (162924) but significantly exceeded that of the clear AF (17 9 11; PB 0.0001) and of the negative control (25 99; PB 0.0001). Checkerboard assay indicated that the turbid AF exhibited a dose-dependent chemotactic activity for PMNs. The turbid AF contained higher levels of TNFa, IL-1b and IL-8 than the clear AF. The concentration of IL-8 in the AF was correlated positively with the chemotactic activity of the AF (r =0.733, P= 0.0005). Anti-human IL-8 antibody added in the turbid AF dose-dependently abolished the chemotactic
* Corresponding author. Tel.: + 81-285-442111; fax: + 81-285-448505. E-mail address: [email protected] (H. Minakami) 0165-0378/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 5 - 0 3 7 8 ( 9 9 ) 0 0 0 4 8 - 0
22
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
activity of the turbid AF. It is concluded that meconium-stained AF is a chemoattractant for PMNs and that cytokines such as an IL-8 may be involved in this phenomenon. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Amniotic fluid; Chemotaxis; Leukocyte; Meconium; Term labour
1. Introduction Staining of the amniotic fluid (AF) with meconium (turbid AF) reportedly occurs in 7–25% of term pregnancies (Katz and Bowes, 1992; Berkus et al., 1994). The adverse effects of meconium on the infant include pulmonary disease, pulmonary hypertension of the newborn, and pneumothorax (Katz and Bowes, 1992; Nathan et al., 1994), while the mother is at an increased risk of postpartum infection (Markovitch et al., 1993; Piper et al., 1998). Reports of any possible beneficial effect of meconium on the infant are limited (Montgomery et al., 1995). In an in vitro study, Montgomery et al. (1995) observed that meconium inhibited the contractile effect of the thromboxane A2 analogue U46619, in the umbilical artery and in venous smooth muscle, which suggests that meconium can neutralize certain vasoconstrictor stimuli in utero. The passage of meconium may result from fetal asphyxia in some patients. If the fetal demand for oxygen exceeds the placental ability to supply it, the fetus is at an increased risk for hypoxia and it may defecate meconium into the AF. In the presence of insufficient placental function, the spontaneous onset of the labour may result in the live birth of the infant. Term parturition is associated with an increased concentration of neutrophil attractant in the AF (Romero et al., 1991). Our hypothesis is that the turbid AF possesses a stronger chemotactic activity for polymorphonuclear leukocytes (PMNs) than the clear AF in term pregnancy.
2. Material and methods
2.1. Study subjects and preparation of amniotic fluid (AF) After obtaining informed consent, specimens of AF were collected during cesarean section performed in 17 women for an obstetrical indication other than fetal distress. Samples were obtained from nine women who underwent an elective cesarean section at 38.091.2 weeks of gestation. Turbid AF were obtained from eight women: four underwent an elective cesarean section at 39.09 1.8 weeks of gestation and had light meconium; four underwent an intrapartum cesarean section for dystocia at 39.090.8 weeks
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
23
of gestation and had thick meconium. In all cases, the maternal serum level of C-reactive protein on the day before the cesarean section was B0.4 mg/dL. On the day of cesarean section, each woman exhibited a normal body temperature. Twelve women who underwent an elective cesarean section had no labour pains. The 17 neonates born to these mothers had no signs of infection and did well during their hospital stay. The mean birthweight of the nine infants with clear AF and of the eight infants with turbid AF did not differ significantly: 29259289 g vs. 30359450 g, respectively. Part of each specimen of AF was cultured for aerobic and anaerobic bacteria, Mycoplasma hominis, Ureaplasma urealyticum, Candida species, and Chlamydia trachomatis. The remainder of each sample was centrifuged at 1500× g for 10 min and the supernatant was used in studies of clear or turbid AF. As the negative control, we used RPMI-1640 (Gibco BRL, USA) and, as the positive control, RPMI-1640 containing 10 nM N-formylmethionyl-leucyl-phenylalanine (fMLP, Peninsula Laboratories, Belmont, USA), which is a known chemoattractant for PMNs (Williams et al., 1977). Samples and controls were stored at −80°C until assayed. Four groups of specimens were prepared: clear AF (n=9), turbid AF (n=8), negative control (n=6), and positive control (n= 6). PMNs were obtained from three healthy volunteer women with uncomplicated singleton pregnancies at term. PMNs were isolated according to the method of Dooley et al. (1982), and were suspended at a concentration of 5×106/ml in RPMI-1640 with 10% fetal calf serum (FCS).
2.2. Chemotactic assay and checkerboard assay The in vitro chemotactic activities of the clear AF in the presence or absence of exogenous IL-8 (recombinant human IL-8; R& D Systems, Minneapolis, USA), the turbid AF in the presence or absence of monoclonal anti-human IL-8 antibody (R& D Systems, Minneapolis, USA), the negative control, and the positive control for PMNs were measured using a modified blind well chamber (Neuro Probe, Washington, DC) employing Boyden’s technique, as described by Ward et al. (1965). The chamber had two compartments separated by a polycarbonate filter, having a pore diameter of 3 mm. An aliquot of 200 ml of the AF or control was placed in the lower compartment, and a 100 ml suspension of PMNs was placed in the upper compartment. After incubation for 40 min at 37°C in 5% CO2 in air, the PMNs that migrated to the lower side of the filter were stained with Diff-Quik (Kokusaisiyaku, Kobe, Japan) and their number in five randomly selected high-power fields (× 400) was counted under a light-microscope. Chemotactic activity was expressed as the number of PMNs counted. All
24
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
assays were performed in duplicate. To confirm the chemotactic activity of turbid AF for PMNs, a checkerboard assay was performed according to the method of Zigmond and Hirsch (1973). Various concentrations of the turbid AF (0–64% in final) were added to the PMN suspension in the upper compartment and to the negative control (RPMI-1640 with 10% FCS) in the lower compartment.
2.3. Assay of TNFa, IL-1b, and IL-8 in AF The concentrations of TNFa, IL-1b, and IL-8 were measured in AF using commercially available kits (Human TNFa Immunoassay, R& D systems, Minneapolis, USA; Human IL-1b (J Immunoassay, R& D systems, Minneapolis, USA; and Human interleukin-8 ELISA kit, Toray-Fuji Bionics, Tokyo, respectively) according to the manufacturer’s instruction. The minimum detectable concentration for TNFa, IL-1b and IL-8 was 0.2, 0.1, and 1.1 ng/l, respectively. The intra- and inter-assay coefficients of variation for each cytokine were B9 and 12%, respectively.
2.4. Statistical analysis Data are reported as mean91 SD. Wilcoxon rank-sum test was used to compare means. Spearman rank correlation coefficients were determined to analyze the linear regression between the number of PMNs migrated and the concentration of TNFa, IL-1b, or IL-8. A level of PB0.05 was accepted as statistically significant.
3. Results Results of bacterial culture were negative in all 17 samples of AF. The Diff-Quik-stained PMNs that migrated from the upper compartment and were trapped on the filter of the lower compartment side are illustrated in Fig. 1. The number of PMNs (200959) that migrated from the upper compartment in which suspension of PMNs was placed to the turbid AF in the lower compartment was comparable to that of the positive control (1629 24), but was significantly higher than that of the clear AF (17911, P B 0.0001) as well as that of the negative control (2599, PB0.0001) (Fig. 2). Although four samples of the eight turbid AF were obtained from women without labour pains, the chemotactic activity for PMNs of these AF samples was comparable with that of the AFs obtained from four women during an intrapartum cesarean section.
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
25
To test the chemotactic activity of turbid AF for PMNs, a checkerboard assay was performed (Fig. 3). In the presence of 64% turbid AF (final concentration) in the lower compartment, 200, 175, 108, and 39 PMNs migrated from the upper compartment in which 0, 4, 16, and 64% turbid AF was present, respectively, to the lower compartment. In the absence of turbid AF in the lower compartment, varying concentrations of turbid AF in the upper compartment did not influence the number of migrated PMNs and only 20–39 PMNs migrated. In the absence of turbid AF in the upper compartment, the number of migrated PMNs increased with increasing concentration of turbid AF in the lower compartment. Thus, the results of the checkerboard assay indicated that the concentration gradient of turbid AF was important in the migration of PMNs, which confirmed that turbid AF possessed a dose-dependent chemotactic activity for PMNs. With a negative gradient of turbid AF, in which the upper compartment contained less turbid AF than the lower compartment, the migration of PMNs from the upper to the lower compartment increased in accordance with an increase in the difference in concentration of turbid AF between the two chambers. In contrast, a positive gradient of turbid AF between the two chambers did not enhance the migration of PMNs.
Fig. 1. Photomicrographs showing polymorphonuclear leukocytes (PMNs) that had migrated to: (a) the negative control; (b) the positive control; (c) clear amniotic fluid; and (d) meconium-stained (turbid) amniotic fluid. Arrows in (a, d) indicate leukocytes. Two leukocytes are observed in (a), while about 50 are present in (d). Filter pores are indicated by arrowheads.
26
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
Fig. 2. Diagram showing the number of polymorhonuclear leukocytes (PMNs) that had migrated to the negative (n= 6) and positive controls (n= 6), clear amniotic fluid (n= 9) and meconium-stained (turbid) amniotic fluid (n= 8). Closed triangles indicate the negative and positive controls. Open circles indicate the AF obtained from women who underwent an elective cesarean section in the absence of labour pains. Closed circles indicate the AF obtained from women who underwent an intrapartum cesarean section.
Concentrations of TNFa, IL-1b and IL-8 were measured in nine samples of clear AF and eight samples of turbid AF. The turbid AF contained higher levels of TNFa, IL-1b and IL-8 than did the clear AF (P B 0.0001) (Fig. 4). No overlap in the concentration of any of these three cytokines was observed between the clear and the turbid AF. Turbid AF obtained during an intrapartum cesarean section tended to contain a higher level of each cytokine than did turbid AF obtained during an elective cesarean section. A strongest significant correlation was observed between the number of PMNs migrated and the concentration of IL-8 in the AF. A significant correlation was observed also between the concentrations of IL-1b and IL-8 in the AF (data not shown; r=0.936, P B 0.0001). To examine whether IL-8 was responsible for the chemotactic activity of turbid AF, varied concentrations of recombinant IL-8 (rIL-8) were added to the clear AF or varied concentrations of anti-human IL-8 antibody were added to the turbid AF. The chemotactic activities of these modified AFs were then examined. Addition of rIL-8 to the clear AF conferred dose-dependent chemotactic activity for PMNs on the clear AF (Fig. 5A). Addition of anti-human IL-8 antibody in the turbid AF dosedependently abolished the chemotactic activity of the turbid AF (Fig. 5B).
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
27
4. Discussion The present study has demonstrated that meconium-stained, turbid, AF exhibits a biological neutrophil attractant activity equal to that of 10 nM fMLP in the absence of bacteria and clinical infection. In such AF that possessed neutrophil attractant activity, levels of the so-called inflammatory cytokines such as TNFa, IL-1b, and IL-8 were markedly increased. Among cytokines examined in the present study, concentration of IL-8 in the AF had the strongest correlation with the chemotactic activity of the AF. IL-8 is released by many uterine tissues, including the placenta (Shimoya et al., 1992), the chorion-decidua (Kelly et al., 1992) and the cervix (Bardlay et al., 1993), and is a potent neutrophil attractant and activator (Baggiolini et al., 1989). IL-8 is reportedly produced by normal colonic epithelial cells (Patel et al., 1995) and was recently reported to be present in meconium, and has
Fig. 3. Effect of differing concentrations of turbid AF between the upper and lower compartments on the number of PMNs that migrated from the upper compartment to the lower compartment. Values represent the mean of duplicate experiments. In the presence of a higher concentration of the turbid AF in the lower compartment than in the upper compartment, a larger number of PMNs migrated from the upper compartment to the lower compartment.
28
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
Fig. 4. Relationships between the number of PMNs migrated and the concentration of TNFa, IL-1b or IL-8 in the AF. Clear AF () (n= 9), turbid AF obtained during an elective cesarean section in the absence of labour pains ( ) (n= 4), and turbid AF obtained during an intrapartum cesarean section ( ) (n= 4). A significant correlation was observed between the number of PMNs migrated and the concentration of IL-1b (r= 0.688, P = 0.0016), between the number of PMNs migrated and the concentration of IL-8 (r= 0.733, P = 0.0005), but not between the number of PMNs migrated and the concentration of TNFa (r=0.471, P =0.055).
been suggested to be responsible for the neutrophil attractant activity of meconium-stained AF (Beaufort et al., 1998). The results of these previous studies and our present findings suggest that turbid AF possesses a neu-
Fig. 5. Effects of exogenous recombinant IL-8 and anti-human IL-8 antibody on the chemotactic activity of AF. (A) Varied concentrations of IL-8 were added to the clear AF before chemotactic activity of the clear AF was assayed. (B) Varied concentrations of anti-human IL-8 antibody were added to the turbid AF before chemotactic activity of the turbid AF was assayed. Vertical bar indicates the mean 9 SD of triplicate experiments.
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
29
trophil attractant activity due to the action of the IL-8, although the exact source of IL-8 is not known. We did not examine histologically the placentas, chorion or uterine cervix of the 17 women studied. However, it has been reported that the presence of neutrophil attractant in AF is a more sensitive marker for histologicallyverified chorioamnionitis and early delivery than is the culture of AF (Cherouny et al., 1993). It is possible that a larger number of PMNs may have invaded these tissues of the women with turbid AF compared to those with clear AF. The PMNs that accumulate around the uterine cervix may play a role in ripening by causing collagenolysis (Junqueira et al., 1980; Rajabi et al., 1988; Osmers et al., 1992). The degree of neutrophil invasion of the cervical connective tissue reportedly parallels the clinical progression of cervical dilatation (Junqueira et al., 1980; Rajabi et al., 1988; Osmers et al., 1992). These findings suggest that the appearance of neutrophil attractant in the AF precedes the onset of frequent uterine contractions and labour pains, even in term labour, via the accumulation of PMNs in the absence of infection. It is not known whether such an accumulation of PMNs is required for the commencement of labour. In conclusion, although the exact source of IL-8 found in turbid AF is not determined, the meconium which contains IL-8 may exhibit neutrophil attractant activity in vivo once it is discharged into the AF. This phenomenon could be related indirectly to the commencement of labour pains.
Acknowledgements The authors are grateful to the Japanese Ministry of Health and Welfare for financial support (Pediatric Research Grant c 10-02).
References Baggiolini, M., Walz, A., Kunkel, S.L., 1989. Nerutrophil-activating peptide-1/interleukin-8, a novel cytokine that activates neutrophils. J. Clin. Invest. 84, 1045 – 1049. Bardlay, C.G., Brennand, J.E., Kelly, R.W., Galder, A.A., 1993. Interleukin-8 production by the human cervix. Am. J. Obstet. Gynecol. 169, 625 – 632. Beaufort, A.J., Pelikan, D.M.V., Elferink, J.G.R., Berger, H.M., 1998. Effect of interleukin 8 in meconium on in vitro neutrophil chemotaxis. Lancet 352, 102 – 105. Berkus, M.D., Langer, O., Samueloff, A., Xenakis, E.M.J., Field, N.T., Ridgeway, L.E, 1994. Meconium-stained amniotic fluid: increased risk for adverse neonatal outcome. Obstet. Gynecol. 84, 115–120.
30
T. Yamada et al. / Journal of Reproducti6e Immunology 46 (2000) 21–30
Cherouny, P.H., Pankuch, G.A., Romero, R., Botti, J.J., Kuhn, D.C., Demers, L.M., Appelbaum, P.C., 1993. Neutrophil attractant/activating peptide-1/interleukin-8: association with histologic chorioamnionitis, preterm delivery, and bioactive amniotic fluid leukoattractants. Am. J. Obstet. Gynecol. 169, 1299 – 1303. Dooley, D.C., Simpson, J.F., Meryman, H.T., 1982. Isolation of large numbers of fully viable human neutrophils: a preparative technique using Percoll density gradient centrifugation. Exp. Hematol. 10, 591–599. Junqueira, L.C.U., Zugaib, M., Montes, G.S., Toledo, O.M.S., Krisztan, R.M., Shigihara, K.M., 1980. Morphological and histological evidence for the occurrence of collagenolysis and for the role of neutrophilic polymorphonuclear leukocytes during cervical ripening. Am. J. Obstet. Gynecol. 138, 273 – 281. Katz, V.L., Bowes, W.A., 1992. Meconium aspiration syndrome: reflections on a murky subject. Am. J. Obstet. Gynecol. 166, 171 – 183. Kelly, R.W., Leask, R., Galder, A.A., 1992. Choriodecidual production of interleukin-8 and mechanism of parturition. Lancet 339, 776 – 777. Markovitch, O., Mazor, M., Shoham-Vardi, I., Chaim, W., Leiberman, J.R., Glezerman, M., 1993. Meconium stained amniotic fluid is associated with maternal infectious morbidity in preterm delivery. Acta Obstet. Gynecol. Scand. 72, 538 – 542. Montgomery, L.D., Belfort, M.A., Saade, G.R., Moise, K.J. Jr., Vedernikov, Y.P., 1995. Meconium inhibits the contraction of umbilical vessels induced by thromboxane A2 analog U46619. Am. J. Obstet. Gynecol. 173, 1075 – 1078. Nathan, L., Leveno, K.J., Carmody, T.J., Kelly, M.A., Sherman, M.L., 1994. Meconium: a 1990s perspective on an old obstetric hazard. Obstet. Gynecol. 83, 329 – 332. Osmers, R., Rath, W., Adelmann-Grill, B.C., Fittkow, C., Kuloczik, M., Szeverenyi, M., 1992. Origin of cervical collagenase during parturition. Am. J. Obstet. Gynecol. 166, 1455–1460. Patel, R.T., Bain, I., Youngs, D., Keighley, M.R.B., 1995. Cytokine production in pouchitis is similar to that in ulcerative colitis. Dis Colon Rectum 38, 831 – 837. Piper, J.M., Newton, E.R., Berkus, M.D., Peairs, W.A., 1998. Meconium: a marker for peripartum infection. Obstet. Gynecol. 91, 741 – 745. Rajabi, M.R., Dean, D.D., Beydoun, S.M., Woessner, J.F., 1988. Elevated tissue levels of collagenase during dilation of uterine cervix in human parturition. Am. J. Obstet. Gynecol. 159, 971–976. Romero, R., Mazor, M., Avia, C., Ceska, M., Lindley, I., 1991. Neutrophil attractant/activating peptide-1/interleukin-8 in term and preterm parturition. Am. J. Obstet. Gynecol. 165, 813–820. Shimoya, K., Matsuzaki, N., Taniguchi, T., Kameda, T., Koyama, M., Neki, R., Saji, F., Tanizawa, O., 1992. Human placenta constitutively produces interleukin-8 during pregnancy and enhances its production in intrauterine infection. Biol. Reprod. 47, 220 – 226. Ward, P.A., Cochrane, C.G., Muller-Eberhard, H.J., 1965. The role of serum complement in chemotaxis of leukocytes in vitro. J. Exp. Med. 122, 327 – 346. Williams, L.T., Snyderman, R., Pike, M.C., Lefkowitz, R.J., 1977. Specific receptor sites for chemotactic peptides on human polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. USA 74, 1204–1208. Zigmond, S.H., Hirsch, J.G., 1973. Leukocyte locomotion and chemotaxis: new methods for evaluation and demonstration of a cell-derived chemotactic factor. J. Exp. Med. 137, 387–410.
.