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Mechanical stretching induces interleukin-8 gene expression in fetal membranes: a possible role for the initiation of human parturition
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European Journal of Obstetrics & Gynecology and Reproductive Biology 70 (1996) 191 196
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Mechanical stretching induces interleukin-8 gene expression in fetal membranes" a possible role for the initiation of human parturition Kayoko Maehara ~'*, Naohiro Kanayama ~, Emad EL. Maradny ~, Tadayoshi Uezato b, Michiya Fujita b, Toshihiko Terao a "Department ~/' Obstetrics and Gynecology, Hamamatsu University School gf Medicine, 3600 Handa-cho, Hamamatsu, 431-31, Japan bDepartment of Biochemistry II, Hamamatsu l'niversitv School g/" ,!4edicine, 3600 Handa-cho, Hamarnatsu, 431-31, Japan Received 29 April 1996; revised 30 July 1996; accepted 6 September 1996
Abstract
Objective: Interleukin-8 (IL-8) is known to play a crucial role in human parturition. We aimed to study the effect of mechanical stretching on the expression of IL-8 in fetal membranes (anmiochorion) and decidua. Study design: We examined the expression of IL-8 and its receptor in fetal membranes (amniochorion) and decidua by immunohistochemical staining. Also, we studied the synthesis of IL-8 messenger RNA (mRNA) in the fetal membranes before and after stretching. Results: We found that mechanical stretching within physiological limit increased IL-8 messenger RNA (mRNA) synthesis in fetal membranes and decidua in a timeand load-dependent manner. Application of mechanical force led to markedly increased staining of IL-8 receptor in decidual cells but not in amnion or chorion cells.Conclusion: These results suggested that mechanical stretching was a candidate for one of the signals important for production of IL-8 in fetal membranes and decidua and probably for initiation of a cytokine network at amniochorio-decidual intert~ace through increased expression of IL-8 receptors. Copyright © 1996 Elsevier Science Ireland Ltd Keywor&': Interleukin-8 (Ik-8); Mechanical stretching: Messenger RNA (mRNA); Fetal membranes; Decidua
1. Introduction
Interleukin-8 (IL-8) is an activator and chemoattractant of neutrophils [1]. Cervix and lower segments of uterine are known to produce large amounts of IL-8 at terminal stages of pregnancy [2,3]. Cultured amnion, chorion, and decidual cells were found to produce IL-8 in response to endotoxin and inflammatory cytokines [4,5]. An exogenous application of IL-8 is known to induce cervical ripening through increased release of collagenolytic enzymes from cervical fibroblasts and accumulated neutrophils before onset of inflammatory response [6,7]. This finding suggests a possible role of IL-8 in initiating parturition. *Corresponding author. Tel.: +81 53 4352309: fax: +81 53 4352308.
Controversy still exists about identification of a mechanism(s) that potentiates the production and release of IL-8 in normal parturition. Most studies have focused on humoral factors and have not attempted to examine a physical factor(s) as a causative agent(s). During parturition the advancing fetal head should rapidly expand fetal membranes and lower uterine segment. Furthermore, mechanical maneuvers such as the stripping of the membranes and the inflation of a balloon would add to straining of these tissues. These considerations have prompted us to investigate the effect of mechanical stretching on IL-8 expression in fetal membranes and decidua obtained at caesarean section. As it is necessary for IL-8 to bind to its receptor to initiate the response in target cells [8,9], we have estimated the expression of IL-8 receptors in these tissues by immunohistochemistry. Here we report that mechanical stretching increased both IL-8 messenger
0301-2115,96,515.00 Copyright ,c' 1996 Elsevier Science Ireland Ltd. All righls reserved PII S(13()1-2115(95)02602-4
192
K. Maehara et al. European Journal qf Obstetrics & Gynecology and Reproductive Biology 70 (1996) 191 196
RNA (mRNA) and its protein product in fetal membranes and decidua in a time- and load-dependent manner.
2. Materials and methods
2.1. Collection of fetal membranes This research was approved by the Research Ethics Committee of Hamamatsu University School of Medicine. A written consent was obtained from each of 14 patients. The fetal membranes including decidua were those from normal term gestations (37 40 weeks) obtained at the time of elective caesarean section before the onset of labour. Samples were washed thoroughly in sterile phosphate-buffered saline (PBS), pH 7.2, to remove adherent blood clots. Membranes were divided into 3 × 5 cm strips. We used three types of membranes: whole membranes (amniochorion and adherent decidua), chorion (chorion and adherent decidua), and amnion. The membranes were stained with hematoxylin and eosin for histological examination.
2.2. Stretching oJ" the fetal membranes We constructed an apparatus according to the modified techniques of Artal et al. [10]. In brief, this device consisted of two pairs of jaws for clamping the membrane samples, a specimen trough, and a spring balance. One pair of jaws bit the sample and the other pair was attached to the spring balance to determine the load-deformation characteristics of the membrane. Force was calculated from the load (g) and the displacement (cm) as the specimen was stretched. The clamped samples were immersed in serum-free minimun essential media (MEM) (Nissui Pharmaceutical, Tokyo) supplemented with 2 mmol glutamine (Nissui Pharmaceutical) at 37°C. We kept the strips stretched for 1, 5 and 12 h under 40 g of tensile force. In similar experiments which lasted for 5 h the loads of 40, 80 and 120 g were applied. Control membranes were under the same conditions except the absence of tensile force. Strips were collected and kept at - 8 0 ° C until further analysis.
the membranes were prehybridized and hybridized with IL-8 complementary DNA (cDNA) probes labelled with [c~-32p]dCTP (Du Pont-New England Nuclear, USA) using a random primer labelling system (Amersham). An IL-8 cDNA probe, a 0.45 kilobase (kb) EcoRI-EcoRI coding region, was obtained from Professor K. Matsushima (Kanazawa University, Japan [l]). After hybridization, the membranes were washed with 2 x standard saline citrate (SSC)/0.1% sodium dodecyl sulfate (SDS) at room temperature and with 0.1 × SSC/0.1% SDS at 65°C and exposed to X-ray film (Fuji, Kanagawa) at - 8 0 ° C for 2 3 days. All blots were rehybridized with beta-actin cDNA probes (Wako, Osaka) to monitor the total mRNA loaded.
2.4. Immunohistochemical staining Jor IL-8 and IL-8 receptor Control and stretched specimens were fixed in 4% paraformaldehyde in PBS and embedded in paraffin wax. Paraffin sections were cut at a thickness of 3-4 /~m. After deparaffinization, the sections were rehydrated with descending grades of alcohol, equilibrated with PBS, pH 7.2, and used for immunohistochemical staining of IL-8 and its receptor (type-l) by a labelled streptavidin-biotin method (LSAB kit; Dako, USA). Briefly, rehydrated sections were quenched in 3% hydrogen peroxide in methanol to block endogenous peroxidase activity, followed by a rinse in PBS solution, and exposed to bovine serum albumin (BSA) PBS solution for 10 min to minimize nonspecific antibody binding. Sections were incubated with rabbit anti-human IL-8 antibody (Upstate Biotechnology, USA) or rabbit anti-human IL-8 receptor antibody (gift from Professor K. Matsushima, Kanazawa University [12]) both at a dilution of 1:100 in PBS solution containing 1% BSA at 4°C for 12 h in a humidified atmosphere. After washing, they were incubated with biotinylated anti-rabbit immunoglobulin at room temperature for 30 min. After further incubation with peroxidase-labelled streptavidin for 30 min, peroxidase activity was located with 3-amino-9-ethylcarbazole. Sections were counterstained with Mayer's hematoxylin. Control sections were subjected to the same procedure except that the primary antibodies were replaced by rabbit non-immune immunoglobulins (Dako).
2.3. RNA extraction and Northern blot analysis RNA extraction and Northern blot analysis were performed as described previously [1,11]. Briefly, strips were homogenized and total cytoplasmic RNA was isolated using guanidinium isothiocyanate. Samples of 10 /~g of total RNA were denatured, separated by electrophoresis in 1% (w/v) agarose gel containing formaldehyde, a n d transferred to Hybond N + membranes (Amersham, USA). After ultraviolet irradiation,
3. Results
3.1. RNA extraction and Northern blot analysis Fig. 1 shows the synthesis of IL-8 mRNA in whole membranes, amnion alone and chorion. Application of 40g load for 12 h induced the synthesis of IL-8 mRNA in all of these tissues. The time-course of its expression
K. Maehara et al. European Journal o! Obstetri(s & Gynecology and Reproductive Biology 70 (1996) 191 196
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/3 -actin B -actin Fig. 1. IL-8 m R N A synthesis in mechanically stretched fetal membranes. Membranes were kept strained under a load of 40 g for 12 h. Total RNA was extracted and hybridized to ~2P-labeled IL-8 and beta-actin cDNA probes. Samples of 10 ~tg of total RNA were loaded. Expression of IL-8 m R N A in: control whole membranes (lane 2), stretched whole membranes (lane 3), control amnion (lane 4), stretched amnion (lane 5), control chorion (lane 6), and stretched chorion (lane 7). IL-8 m R N A induced by A23187 in HL60 cells was used as a positive control (lane 1) [11].
was examined with whole membranes and chorion. There was no difference in the IL-8 m R N A synthesis between 1 and 5 h periods (Fig. 2A, B), lanes 1 4). However, when the duration of the stretching was extended to 12 h a marked increase in the IL-8 m R N A synthesis was observed (Fig. 2A, B), lanes 5 and 6). These findings were reproducible. The load-dependence of IL-8 synthesis was examined with the whole membranes. A load of 40 g for 5 h did not stimulate the synthesis of IL-8 m R N A (Fig. 3, lanes 1 and 2) but application of 80 and 120 g load increased its synthesis with increasing effect (Fig. 3, lanes 3-6). Thus, mechanical stretching stimulated IL-8 m R N A synthesis in the whole membranes in a time- and loaddependent manner.
Fig. 3. Load-dependence of stretching on IL-8 m R N A expression in whole membranes. The membranes were stretched for 5 h with 40, 80 and 120 g load. Northern blot analysis was performed in the same manner as in Fig. 1. Expression of IL-8 m R N A in: control (lane 1, 3. 5), 40 g (lane 2), 80 g (lane 4) and 120 g load (lane 6).
3.2. Immunohistochemical staining jor IL-8 and its receptor Fig. 4 shows representative staining patterns of IL-8 in fetal membranes (amniochorion) and decidua. A weak immunostaining for IL-8 was seen in some chorion cells (Fig. 4A) and in the blood vessels in decidua (Fig. 4C), whereas amnion cells and the compact layer below amniotic epithelium were not stained in the unstretched membranes (Fig. 4A). Alter application of 120 g load for 5 h, immunostaining for IL-8 was much increased in the cytoplasm of amnion and chorion cells (Fig. 4B). The compact layer below amniotic epithelium was also more intensively stained in the stretched membranes than that of the unstretched sample. The reticular layer of chorion and decidual cells showed a strong staining for IL-8 after application of the tensile force (Fig. 4D). Rabbit nonimmune immunoglobulins used as the control yielded no specific staining (data not
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Fig. 2. Time-course of IL-8 m R N A expression induced by mechanical stretching. Whole membranes (A) and chorion (B) were stretched with 40 g load for 1, 5 and 12 h. Northern blot analysis was performed in the same manner as in Fig. 1. Expression of IL-8 m R N A in: 1 h control (lane 1): 1 h stretching (lane 2); 5 h control (hme 3): 5 h stretching (lane 4): 12 h control (lane 5): 12 h stretching (lane 6).
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K. Maehara et al. European Journal qf Obstetrics & @'necology and Reproductiz,e Biology 70 (1996) 191 196
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Fig. 4. lmmunohistochemical staining for IL-8 expression in t'etal membranes (amniochorion) and decidua induced by increased tension. (A) amniochorion section of control membranes; (B) stretched membranes showing strong staining for IL-8 in the cytoplasm of amnion and chorion cells and in the compact layer below amniotic epithelium; (C) decidua section of control membranes: (D) stretched membranes showing strong staining for IL-8 in decidual cells and in the reticular layer of" chorion. Original magnilication, x 100.
shown). Staining for IL-8 of the four other sections showed similar patterns. There was a remarkable difference in the IL-8 staining between the membranes on which 120 g load was applied for 5 h and those of controls. Staining for the IL-8 receptor was positive only for some of the decidual cells in unstretched membranes (Fig. 5C). Application of 120 g load for 5 h had a negligible effect on the staining for this receptor in both amnion and chorion cells (Fig. 5A, B). However, a 120 g 5 h stretching, markedly increased its staining in decidual cells (Fig. 5C, D). Sections from the two other stretched membranes produced similar results.
4. Discussion The major finding of this study was a stimulatory effect of mechanical stretching on IL-8 expression in fetal membranes and decidua. This result was supported by that of the immunohistochemical staining. Many studies have shown that bacterial infection could lead to increased cytokine and prostaglandin levels in fetal membranes and decidua in women suffering from intraamniotic infection [13 16]. However, the signals that trigger the production of cytokines in these
tissues in normal parturition remain to be identified. The decrease of progesterone level was suggested to be one of the stimuli which induce IL-8 production [17,18]. In the present paper, we propose that one of the unidentified factors could be mechanical stretching. We have previously reported that mechanical stretching of cultured amnion cells led to increased synthesis and release of prostaglandin E, [19], a factor related to cervical maturation. Those previous findings of ours seem to lend support to the present results. The tension produced by the load per unit width of membrane is 13 40 g/cm, i.e. 10 30 mmHg/cm. Previous investigators measured the intrauterine pressure with the internal and external recording techniques and expressed it in mmHg. The tonus (the resting state) is the lowest value obtained between contractions and it ranges between 8 and 10 mmHg at term [20]. When the pregnant woman is standing, the lowest part of the uterine walls receives the hydrostatic pressure of the fluid column above it. This pressure is about 20 mmHg which should be added to the pressure exerted by the tonus [20]. Although in this paper we stretched the membrane in only one direction, which might be unphysiological, the tensile strength applied was within the physiological force produced during the fetal growth.
K. Moehara et al. European Journal o/ Ol~s:elri~ & Gynecology amt RG,'oduclire Biolog, 70 (1996) 191 196
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Fig. 5. Immunohistochemical staining for IL-8 receptor after continuous strelching of fetal membranes amniochorion) and decidua. (A) amniochorion section of control membranes: (B) stretched membranes showillg no slain ing for I L-8 receptor at either anmion cells or chorion cells: (C) decidua section of control membranes: (DI stretched membranes showing strong staining for I L-8 receplor at decidual cells. Original magnilication, x 10(I.
Stimulatory effect of stretching on gene expression has been reported in several instances. Mechanical stretching of cardiocyte induced the expression of angiotensinogen [21], c-/os and fetal type skeletal proteins such as alpha actin and beta myosin heavy chain [22]. Cyclical strain induced endothelin-1 [23] and shear stress adhesion molecules in the umbilical endothelial cells [24]. Another finding of this paper was that the stretchinginduced expression of IL-8 receptor was only detected in the decidual tissue in immunohistochemical staining. Although we could not demonstrate the increase of IL-8 receptor mRNA, the above observation suggested that the target cells for I L-8 were mainly located in decidua, a rich source of cytokines and prostaglandins [14,25]. Until now there have been no reports on the interaction between IL-8 and its receptor in fetal membranes and decidua. In this paper, we showed the different distribution of the ligand and the receptor in those membranes which might suggest a possible intercommunication at molecular level between the tissues derived from the mother and the fetus. The present work did not deal with the signal transduction pathways of I[-8 gene expression induced by
stretching. It was reported that mechanical force applied to skeletal muscle fibers induced the rapid increase of the intracellular calcium concentration [26]. Another group also reported that mechanical stress activated a protein kinase-catalyzed cascade of phosphorylation in neonatal rat cardiac myocytes [27].
Acknowledgements We thank Drs T. Oda, and C. Uchida for advice on Northern blot analysis and Professor K. Matsushima (Kanazawa University) for providing IL-8 cDNA probes and IL-8 receptor antibody. This study was supported in part by a Grant-in-Aid for Specific Research (no. 06454469) from the Ministry of Education, Science and Culture of Japan and Adeza Biomedical grant (USA).
References I1] Matsushima K, Morishita K, Yoshimura T, Lavu S. Kobayashi 5". Le~ W. Appella E, Ktmg HF, Leonard EJ, Oppenheim JJ. Molecular cloning of a human monocyte-derived neutrophil
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K. Maehara el al. ,; European Journal o! Obstetrics & Gynecology and Reproductive Biology 70 (1996) 191 196 chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J Exp Med 1988; 167: 1883--1893. Osmers RGW, Blaser J, Kuhn W, Tschesche H. lnterleukin-8 synthesis and the onset of labor. Obstet Gynecol 1995; 86: 223- 229. Barclay CG~ Brennand JE, Kelly RW, Calder AA. lnterleukin-8 production by the human cervix. Am J Obstet Gynecol 1993; 169: 625-632. Kelly RW, Leask R, Calder AA. Choriodecidual production of interleukin-8 and mechanism of parturition. Lancet 1992: 339: 776 777. Dudley DJ, Trautman MS, Mitchell MD. Inflammatory mediators regulate interleukin-8 production by cultured gestational tissues: evidence for a cytokine network at the chorio-decidual interface. J Clin Endocrinol Metab 1993; 76:404 410. El Maradny E, Kanayama N, Halim A, Maehara K, Sumimoto K, Terao T. Interleukin-8 induces cervical ripening in rabbits. Am J Obstet Gynecol 1994: 171:77 83. El Maradny E, Kanayama N, Halim A, Maehara K, Sumimoto K, Terao T. Effects of neutrophil chemotactic factors on cervical ripening. Clin Exp Obst Gyn 1995; 22: 76-85. Holmes WE, Lee J, Kuang W J, Rice GC, Wood WI. Structure and functional expression of a human interleukin-8 receptor. Science 1991; 253:1278 1280. Murphy PM, Tiffany HL. Cloning of complementary DNA encoding a functional human interleukin-8 receptor. Science 1991; 253: 1280-1283. Artal R, Sokol RJ, Neuman M, Burstein AH, Stojkov J. The mechanical properties of prematurely and non-prematurely ruptured membranes. Am J Obstet Gynecot 1976: 125:655 659. Maehara K, Kanayama N, Halim A, El Maradny E, Oda T, Fujita M, Terao T. Down-regulation of interleukin-8 gene expression in HL60 cell line by human Kunitz-type trypsin inhibitor. Biochem Biophys Res Commun 1995: 206:927 934. Morohashi H, Miyawaki T, Nomura H, Kuno K, Murakami S, Matsushima K, Mukaida N. Expression of both types of human interleukin-8 receptors on mature neutrophils, monocytes, and natural killer cells. J Leukoc Biol 1995; 57:180 187. Menon R, Swan KF, Lyden TW, Rote NS, Fortunato SJ. Expression of inflammatory cytokines (interleukin-Ifl and interleukin-6) in amniochorionic membranes. Am J Obstet Gynecol 1995: 172:493 500. Romero R, Wu YK, Brody DT, Oyarzun E, Duff GW, Durum SK. Human decidua: a source of interleukin-1. Obstet Gynecol 1989; 73: 31- 34. Simoya K, Matsuzaki N, Taniguchi T, Kameda T, Koyama M, Neki R, Saji F. Tanizawa O. Human placenta constitutively
produces interleukin-8 during pregnancy and enhances its production in intrauterine infection. Biol Reprod 1992; 47: 220-226. [16] Lamont RF, Rose M, Elder MG. Effect of bacterial products on prostaglandin E production by amnion cells. Lancet 1985; 2: I331 1333. [17] lto A, lmada K, Sato T, Kubo T, Matsushima K, Mori Y. Suppression of interleukin 8 production by progesterone in rabbit uterine cervix. Biochem J 1994; 301: 183- 186. [18] Kelly RW, lllingworth P, Baldie G, Leask R, Brouwer S, Calder AA. Progesterone control of interleukin-8 production in endometrium and chorio-decidual cells underlines the role of the neutrophil in menstruation and parturition. Hum Reprod 1994, 9:253 258. [19] Kanayama N and Fukamizu H. Mechanical stretching increases prostaglandin E 2 in cultured human amnion cells. Gynecol Obstet Invest 1989, 28:123 126. [20] Cibils LA. In: Cibils L.A., ed. Electronic Fetal-Maternal Monitoring. Antepartum/Intrapartum. The Haugue: Martinus Nijhoff. 198l; 1 495. [21] Shyu KG, Chen J J, Shih NL, Chang H, Wang DL, Lien WP, Liew CC. Angiotensinogen gene expression is induced by cyclical mechanical stretch in cultured rat cardiomyocytes. Biochem Biophys Res Commun 1995; 211:241 248. [22] Yamazaki T, Shiojima 1, Komuro I, Nagai R, Yazaki Y. Involvement of the renin-angiotensin system in the development of left ventricular hypertrophy and dysfunction. J Hypertension (Suppl.) 1994; 12:S153 S157. [23] Wang DL, Tang CC, Wung BS, Chen HH, Hung MS, Wang JJ. Cyclical strain increases endothelin-I secretion and gene expression in human endothelial cells. Biochem Biophys Res Commun 1993; 195:1050 1056. [24] Nagel T. Resnick N, Atkinson WL Dewey Jr CF, Gimbrone Jr MA. Shear stress selectively upregulates intercellular adhesion molecule-1 expression in cultured human vascular endothelial cells. J Clin Invest 1994: 94:885 891. [25] Norwitz ER, Bernal AL, Starkey PM. Tumor necrosis factor-:~ selectively stimulates prostaglandin F2~ production by macrophages in human term decidua. Am J Obstet Gynecol 1992; 167:815 820. [26] Escobar AL, Monck JR, Fernandez JM, Vergara JL. Localization of the site of Ca 2 ~ release at the level of a single sarcomere in skeletal muscle fibers. Nature 1994: 367:739 74i. [27] Yamazaki T, Komuro 1, Kudoh S, Zou Y, Shiojima I, Mizuno T, Takano H, Hiroi Y, Ueki K, Tobe K, Kadowaki T, Nagai R, Yazaki Y. Mechanical stress actives protein kinase cascade of phosphorylation in neonatal rat cardiac myocytes. J Clin Invest 1995; 96:438 446.
European Journal of Obstetrics & Gynecology and Reproductive Biology 70 (1996) 191 196
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Mechanical stretching induces interleukin-8 gene expression in fetal membranes" a possible role for the initiation of human parturition Kayoko Maehara ~'*, Naohiro Kanayama ~, Emad EL. Maradny ~, Tadayoshi Uezato b, Michiya Fujita b, Toshihiko Terao a "Department ~/' Obstetrics and Gynecology, Hamamatsu University School gf Medicine, 3600 Handa-cho, Hamamatsu, 431-31, Japan bDepartment of Biochemistry II, Hamamatsu l'niversitv School g/" ,!4edicine, 3600 Handa-cho, Hamarnatsu, 431-31, Japan Received 29 April 1996; revised 30 July 1996; accepted 6 September 1996
Abstract
Objective: Interleukin-8 (IL-8) is known to play a crucial role in human parturition. We aimed to study the effect of mechanical stretching on the expression of IL-8 in fetal membranes (anmiochorion) and decidua. Study design: We examined the expression of IL-8 and its receptor in fetal membranes (amniochorion) and decidua by immunohistochemical staining. Also, we studied the synthesis of IL-8 messenger RNA (mRNA) in the fetal membranes before and after stretching. Results: We found that mechanical stretching within physiological limit increased IL-8 messenger RNA (mRNA) synthesis in fetal membranes and decidua in a timeand load-dependent manner. Application of mechanical force led to markedly increased staining of IL-8 receptor in decidual cells but not in amnion or chorion cells.Conclusion: These results suggested that mechanical stretching was a candidate for one of the signals important for production of IL-8 in fetal membranes and decidua and probably for initiation of a cytokine network at amniochorio-decidual intert~ace through increased expression of IL-8 receptors. Copyright © 1996 Elsevier Science Ireland Ltd Keywor&': Interleukin-8 (Ik-8); Mechanical stretching: Messenger RNA (mRNA); Fetal membranes; Decidua
1. Introduction
Interleukin-8 (IL-8) is an activator and chemoattractant of neutrophils [1]. Cervix and lower segments of uterine are known to produce large amounts of IL-8 at terminal stages of pregnancy [2,3]. Cultured amnion, chorion, and decidual cells were found to produce IL-8 in response to endotoxin and inflammatory cytokines [4,5]. An exogenous application of IL-8 is known to induce cervical ripening through increased release of collagenolytic enzymes from cervical fibroblasts and accumulated neutrophils before onset of inflammatory response [6,7]. This finding suggests a possible role of IL-8 in initiating parturition. *Corresponding author. Tel.: +81 53 4352309: fax: +81 53 4352308.
Controversy still exists about identification of a mechanism(s) that potentiates the production and release of IL-8 in normal parturition. Most studies have focused on humoral factors and have not attempted to examine a physical factor(s) as a causative agent(s). During parturition the advancing fetal head should rapidly expand fetal membranes and lower uterine segment. Furthermore, mechanical maneuvers such as the stripping of the membranes and the inflation of a balloon would add to straining of these tissues. These considerations have prompted us to investigate the effect of mechanical stretching on IL-8 expression in fetal membranes and decidua obtained at caesarean section. As it is necessary for IL-8 to bind to its receptor to initiate the response in target cells [8,9], we have estimated the expression of IL-8 receptors in these tissues by immunohistochemistry. Here we report that mechanical stretching increased both IL-8 messenger
0301-2115,96,515.00 Copyright ,c' 1996 Elsevier Science Ireland Ltd. All righls reserved PII S(13()1-2115(95)02602-4
192
K. Maehara et al. European Journal qf Obstetrics & Gynecology and Reproductive Biology 70 (1996) 191 196
RNA (mRNA) and its protein product in fetal membranes and decidua in a time- and load-dependent manner.
2. Materials and methods
2.1. Collection of fetal membranes This research was approved by the Research Ethics Committee of Hamamatsu University School of Medicine. A written consent was obtained from each of 14 patients. The fetal membranes including decidua were those from normal term gestations (37 40 weeks) obtained at the time of elective caesarean section before the onset of labour. Samples were washed thoroughly in sterile phosphate-buffered saline (PBS), pH 7.2, to remove adherent blood clots. Membranes were divided into 3 × 5 cm strips. We used three types of membranes: whole membranes (amniochorion and adherent decidua), chorion (chorion and adherent decidua), and amnion. The membranes were stained with hematoxylin and eosin for histological examination.
2.2. Stretching oJ" the fetal membranes We constructed an apparatus according to the modified techniques of Artal et al. [10]. In brief, this device consisted of two pairs of jaws for clamping the membrane samples, a specimen trough, and a spring balance. One pair of jaws bit the sample and the other pair was attached to the spring balance to determine the load-deformation characteristics of the membrane. Force was calculated from the load (g) and the displacement (cm) as the specimen was stretched. The clamped samples were immersed in serum-free minimun essential media (MEM) (Nissui Pharmaceutical, Tokyo) supplemented with 2 mmol glutamine (Nissui Pharmaceutical) at 37°C. We kept the strips stretched for 1, 5 and 12 h under 40 g of tensile force. In similar experiments which lasted for 5 h the loads of 40, 80 and 120 g were applied. Control membranes were under the same conditions except the absence of tensile force. Strips were collected and kept at - 8 0 ° C until further analysis.
the membranes were prehybridized and hybridized with IL-8 complementary DNA (cDNA) probes labelled with [c~-32p]dCTP (Du Pont-New England Nuclear, USA) using a random primer labelling system (Amersham). An IL-8 cDNA probe, a 0.45 kilobase (kb) EcoRI-EcoRI coding region, was obtained from Professor K. Matsushima (Kanazawa University, Japan [l]). After hybridization, the membranes were washed with 2 x standard saline citrate (SSC)/0.1% sodium dodecyl sulfate (SDS) at room temperature and with 0.1 × SSC/0.1% SDS at 65°C and exposed to X-ray film (Fuji, Kanagawa) at - 8 0 ° C for 2 3 days. All blots were rehybridized with beta-actin cDNA probes (Wako, Osaka) to monitor the total mRNA loaded.
2.4. Immunohistochemical staining Jor IL-8 and IL-8 receptor Control and stretched specimens were fixed in 4% paraformaldehyde in PBS and embedded in paraffin wax. Paraffin sections were cut at a thickness of 3-4 /~m. After deparaffinization, the sections were rehydrated with descending grades of alcohol, equilibrated with PBS, pH 7.2, and used for immunohistochemical staining of IL-8 and its receptor (type-l) by a labelled streptavidin-biotin method (LSAB kit; Dako, USA). Briefly, rehydrated sections were quenched in 3% hydrogen peroxide in methanol to block endogenous peroxidase activity, followed by a rinse in PBS solution, and exposed to bovine serum albumin (BSA) PBS solution for 10 min to minimize nonspecific antibody binding. Sections were incubated with rabbit anti-human IL-8 antibody (Upstate Biotechnology, USA) or rabbit anti-human IL-8 receptor antibody (gift from Professor K. Matsushima, Kanazawa University [12]) both at a dilution of 1:100 in PBS solution containing 1% BSA at 4°C for 12 h in a humidified atmosphere. After washing, they were incubated with biotinylated anti-rabbit immunoglobulin at room temperature for 30 min. After further incubation with peroxidase-labelled streptavidin for 30 min, peroxidase activity was located with 3-amino-9-ethylcarbazole. Sections were counterstained with Mayer's hematoxylin. Control sections were subjected to the same procedure except that the primary antibodies were replaced by rabbit non-immune immunoglobulins (Dako).
2.3. RNA extraction and Northern blot analysis RNA extraction and Northern blot analysis were performed as described previously [1,11]. Briefly, strips were homogenized and total cytoplasmic RNA was isolated using guanidinium isothiocyanate. Samples of 10 /~g of total RNA were denatured, separated by electrophoresis in 1% (w/v) agarose gel containing formaldehyde, a n d transferred to Hybond N + membranes (Amersham, USA). After ultraviolet irradiation,
3. Results
3.1. RNA extraction and Northern blot analysis Fig. 1 shows the synthesis of IL-8 mRNA in whole membranes, amnion alone and chorion. Application of 40g load for 12 h induced the synthesis of IL-8 mRNA in all of these tissues. The time-course of its expression
K. Maehara et al. European Journal o! Obstetri(s & Gynecology and Reproductive Biology 70 (1996) 191 196
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11_-8
/3 -actin B -actin Fig. 1. IL-8 m R N A synthesis in mechanically stretched fetal membranes. Membranes were kept strained under a load of 40 g for 12 h. Total RNA was extracted and hybridized to ~2P-labeled IL-8 and beta-actin cDNA probes. Samples of 10 ~tg of total RNA were loaded. Expression of IL-8 m R N A in: control whole membranes (lane 2), stretched whole membranes (lane 3), control amnion (lane 4), stretched amnion (lane 5), control chorion (lane 6), and stretched chorion (lane 7). IL-8 m R N A induced by A23187 in HL60 cells was used as a positive control (lane 1) [11].
was examined with whole membranes and chorion. There was no difference in the IL-8 m R N A synthesis between 1 and 5 h periods (Fig. 2A, B), lanes 1 4). However, when the duration of the stretching was extended to 12 h a marked increase in the IL-8 m R N A synthesis was observed (Fig. 2A, B), lanes 5 and 6). These findings were reproducible. The load-dependence of IL-8 synthesis was examined with the whole membranes. A load of 40 g for 5 h did not stimulate the synthesis of IL-8 m R N A (Fig. 3, lanes 1 and 2) but application of 80 and 120 g load increased its synthesis with increasing effect (Fig. 3, lanes 3-6). Thus, mechanical stretching stimulated IL-8 m R N A synthesis in the whole membranes in a time- and loaddependent manner.
Fig. 3. Load-dependence of stretching on IL-8 m R N A expression in whole membranes. The membranes were stretched for 5 h with 40, 80 and 120 g load. Northern blot analysis was performed in the same manner as in Fig. 1. Expression of IL-8 m R N A in: control (lane 1, 3. 5), 40 g (lane 2), 80 g (lane 4) and 120 g load (lane 6).
3.2. Immunohistochemical staining jor IL-8 and its receptor Fig. 4 shows representative staining patterns of IL-8 in fetal membranes (amniochorion) and decidua. A weak immunostaining for IL-8 was seen in some chorion cells (Fig. 4A) and in the blood vessels in decidua (Fig. 4C), whereas amnion cells and the compact layer below amniotic epithelium were not stained in the unstretched membranes (Fig. 4A). Alter application of 120 g load for 5 h, immunostaining for IL-8 was much increased in the cytoplasm of amnion and chorion cells (Fig. 4B). The compact layer below amniotic epithelium was also more intensively stained in the stretched membranes than that of the unstretched sample. The reticular layer of chorion and decidual cells showed a strong staining for IL-8 after application of the tensile force (Fig. 4D). Rabbit nonimmune immunoglobulins used as the control yielded no specific staining (data not
B
A IL-8
12
34
56 IL-8
m~ .
B-actin
1 23456
-----
.
.
.
/3-actin
Fig. 2. Time-course of IL-8 m R N A expression induced by mechanical stretching. Whole membranes (A) and chorion (B) were stretched with 40 g load for 1, 5 and 12 h. Northern blot analysis was performed in the same manner as in Fig. 1. Expression of IL-8 m R N A in: 1 h control (lane 1): 1 h stretching (lane 2); 5 h control (hme 3): 5 h stretching (lane 4): 12 h control (lane 5): 12 h stretching (lane 6).
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K. Maehara et al. European Journal qf Obstetrics & @'necology and Reproductiz,e Biology 70 (1996) 191 196
A
Fig. 4. lmmunohistochemical staining for IL-8 expression in t'etal membranes (amniochorion) and decidua induced by increased tension. (A) amniochorion section of control membranes; (B) stretched membranes showing strong staining for IL-8 in the cytoplasm of amnion and chorion cells and in the compact layer below amniotic epithelium; (C) decidua section of control membranes: (D) stretched membranes showing strong staining for IL-8 in decidual cells and in the reticular layer of" chorion. Original magnilication, x 100.
shown). Staining for IL-8 of the four other sections showed similar patterns. There was a remarkable difference in the IL-8 staining between the membranes on which 120 g load was applied for 5 h and those of controls. Staining for the IL-8 receptor was positive only for some of the decidual cells in unstretched membranes (Fig. 5C). Application of 120 g load for 5 h had a negligible effect on the staining for this receptor in both amnion and chorion cells (Fig. 5A, B). However, a 120 g 5 h stretching, markedly increased its staining in decidual cells (Fig. 5C, D). Sections from the two other stretched membranes produced similar results.
4. Discussion The major finding of this study was a stimulatory effect of mechanical stretching on IL-8 expression in fetal membranes and decidua. This result was supported by that of the immunohistochemical staining. Many studies have shown that bacterial infection could lead to increased cytokine and prostaglandin levels in fetal membranes and decidua in women suffering from intraamniotic infection [13 16]. However, the signals that trigger the production of cytokines in these
tissues in normal parturition remain to be identified. The decrease of progesterone level was suggested to be one of the stimuli which induce IL-8 production [17,18]. In the present paper, we propose that one of the unidentified factors could be mechanical stretching. We have previously reported that mechanical stretching of cultured amnion cells led to increased synthesis and release of prostaglandin E, [19], a factor related to cervical maturation. Those previous findings of ours seem to lend support to the present results. The tension produced by the load per unit width of membrane is 13 40 g/cm, i.e. 10 30 mmHg/cm. Previous investigators measured the intrauterine pressure with the internal and external recording techniques and expressed it in mmHg. The tonus (the resting state) is the lowest value obtained between contractions and it ranges between 8 and 10 mmHg at term [20]. When the pregnant woman is standing, the lowest part of the uterine walls receives the hydrostatic pressure of the fluid column above it. This pressure is about 20 mmHg which should be added to the pressure exerted by the tonus [20]. Although in this paper we stretched the membrane in only one direction, which might be unphysiological, the tensile strength applied was within the physiological force produced during the fetal growth.
K. Moehara et al. European Journal o/ Ol~s:elri~ & Gynecology amt RG,'oduclire Biolog, 70 (1996) 191 196
A
195
B
Fig. 5. Immunohistochemical staining for IL-8 receptor after continuous strelching of fetal membranes amniochorion) and decidua. (A) amniochorion section of control membranes: (B) stretched membranes showillg no slain ing for I L-8 receptor at either anmion cells or chorion cells: (C) decidua section of control membranes: (DI stretched membranes showing strong staining for I L-8 receplor at decidual cells. Original magnilication, x 10(I.
Stimulatory effect of stretching on gene expression has been reported in several instances. Mechanical stretching of cardiocyte induced the expression of angiotensinogen [21], c-/os and fetal type skeletal proteins such as alpha actin and beta myosin heavy chain [22]. Cyclical strain induced endothelin-1 [23] and shear stress adhesion molecules in the umbilical endothelial cells [24]. Another finding of this paper was that the stretchinginduced expression of IL-8 receptor was only detected in the decidual tissue in immunohistochemical staining. Although we could not demonstrate the increase of IL-8 receptor mRNA, the above observation suggested that the target cells for I L-8 were mainly located in decidua, a rich source of cytokines and prostaglandins [14,25]. Until now there have been no reports on the interaction between IL-8 and its receptor in fetal membranes and decidua. In this paper, we showed the different distribution of the ligand and the receptor in those membranes which might suggest a possible intercommunication at molecular level between the tissues derived from the mother and the fetus. The present work did not deal with the signal transduction pathways of I[-8 gene expression induced by
stretching. It was reported that mechanical force applied to skeletal muscle fibers induced the rapid increase of the intracellular calcium concentration [26]. Another group also reported that mechanical stress activated a protein kinase-catalyzed cascade of phosphorylation in neonatal rat cardiac myocytes [27].
Acknowledgements We thank Drs T. Oda, and C. Uchida for advice on Northern blot analysis and Professor K. Matsushima (Kanazawa University) for providing IL-8 cDNA probes and IL-8 receptor antibody. This study was supported in part by a Grant-in-Aid for Specific Research (no. 06454469) from the Ministry of Education, Science and Culture of Japan and Adeza Biomedical grant (USA).
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