Mol. Hum. Reprod. Advance Access originally published online on June 11, 2004
Molecular Human Reproduction 2004 10(8):573-580; doi:10.1093/molehr/gah077
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Cyclic mechanical stretch augments both interleukin-8 and monocyte chemotactic protein-3 production in the cultured human uterine cervical fibroblast cells*
1Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507 and 2Research & Development Division, Nippon Organon K.K., 5-90 Tomobuchi-cho 1-chome, Miyakojima-ku, Osaka 534-0016, Japan
1 To whom correspondence should be addressed. Email: ihiroaki{at}kuhp.kyoto-u.ac.jp
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Abstract |
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Intensive local leukocyte infiltration in the uterine cervix is a characteristic feature in the process of cervical ripening. The infiltrated leukocytes include neutrophils, macrophages and monocytes, which are believed to play important roles in cervical ripening by secreting elastase, matrix metalloproteinase and interleukin-1 (IL-1). Interleukin-8 (IL-8) and monocyte chemotactic protein-3 (MCP-3) belong to the CXC and CC chemokine families, and mediate the chemotaxis of neutrophils and monocytes/macrophages respectively. The aim of the present study was to investigate the possible involvement of IL-8 and MCP-3 in leukocyte chemotaxis in cervical ripening. Immunohistochemistry and RT–PCR detected both IL-8 and MCP-3 expression in human pregnant uterine cervices. Labour-like cyclic mechanical stretch for 48 h significantly elevated both IL-8 (555%) and MCP-3 (360%) secretion from cultured human uterine cervical fibroblast (CxF) cells (P<0.05 for both). Cyclic mechanical stretch for 24, 36 and 48 h significantly increased both IL-8 and MCP-3 mRNA expression in CxF cells (P<0.05 for all). The stretch-induced augmentation of both IL-8 and MCP-3 expression was significantly suppressed by an activator protein-1 (AP-1) inhibitor, curcumin. These data suggest that cyclic mechanical stretch of the uterine cervix by the presenting part of the fetus during labour may augment both IL-8 and MCP-3 production in the uterine cervix via AP-1 activation.
Key words: cervical ripening/interleukin-8/mechanical stretch/monocyte chemotactic protein-3/uterine cervix
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Introduction |
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During pregnancy, the uterine cervical canal remains tightly closed until the onset of labour, although the uterine corpus cavity expands gradually to accommodate the growing fetus. After the onset of active labour with vigorous contraction of the uterine corpus, rapid ripening and dilatation of the uterine cervix usually commence, followed by the delivery of the conceptus (Cunningham et al., 2001
; Tenore, 2003).
Ripening of the uterine cervix is characterized by two distinct biochemical phenomena, intensive local leukocyte infiltration and rapid degradation of extracellular matrix proteins (Junqueira et al., 1980; Liggins, 1981; Kelly, 1994). In this regard, Liggins (1981) first demonstrated that cervical ripening is an inflammatory reaction. Among various changes in extracellular matrix proteins in cervical ripening, degradation of collagens by the action of metalloproteinases (MMP) is hypothesized to play crucial roles (Osmers et al., 1995a; Sato et al., 1996; Imada et al., 1997; Ledingham et al., 1999). Recently, we have reported that inflammation-associated bioactive substances, such as nitric oxide, inflammatory cytokines, and prostaglandin F2
, as well as a physical force, cyclic mechanical stretch, augment MMP-1 production in cultured human uterine cervical fibroblast cells prepared from pregnant women, thereby suggesting a possible involvement of MMP-1 production by cervical fibroblast cells in the degradation of collagen fibrils at the time of cervical ripening (Yoshida et al., 2001, 2002).
The local infiltration of a considerable number of leukocytes was reported in the uterine cervix in the process of cervical ripening (Junqueira et al., 1980; Liggins, 1981; Kelly, 1994). The major population of infiltrated leukocytes in the human cervix in labour is neutrophils (Junqueira et al., 1980; Kelly, 1994). It was demonstrated that such neutrophils cause degranulation and release elastase (Uldbjerg et al., 1983a; Kanayama and Terao, 1991) and MMP-8 (Osmers et al., 1995b; Winkler et al., 1999a,b), both of which directly degrade collagen fibrils in the cervix (Uldbjerg et al., 1983a,b; Kanayama and Terao, 1991; Osmers et al., 1995b; Winkler et al., 1999a,b). On the other hand, monocyte/macrophage infiltration was also demonstrated in the human cervix at parturition (Bokstrom et al., 1997), although the population of monocytes/macrophages is rather small compared to that of neutrophils. It has been demonstrated that pro-inflammatory cytokines, such as interleukin-1 (IL-1), tumour necrosis factor- (TNF-) and interleukin-6 (IL-6), are mainly produced by monocytes/macrophages (Cavaillon, 1994). These proinflammatory cytokines play key roles in the process of cervical ripening by stimulating the production of various kinds of bioactive substances such as nitric oxide, prostaglandins and MMP (Ito et al., 1987, 1991; Chwalisz et al., 1994; Kelly, 1994; Chwalisz and Garfield, 1998; Norman et al., 1998; Watari et al., 1999; Yoshida et al., 2001, 2002). Thus, it is plausible that the infiltration of both neutrophils and monocytes/macrophages contributes to the degradation of extracellular matrix proteins in the complicated process of cervical ripening. However, the mechanism of regulation of leukocyte infiltration at the time of delivery has not been fully clarified.
Cervical ripening usually proceeds in parallel with the progression of labour, involving the cyclic contraction of the uterine corpus (Cunningham et al., 2001). Braxton Hicks uterine contractions as well as active labour cause cyclic descent of the presenting part of the fetus, resulting in cyclic stretch of the uterine cervix in parturition (Cunningham et al., 2001). However, it remains to be elucidated whether cyclic mechanical stretch directly initiates chemotaxis of neutrophils and/or monocytes/macrophages in the process of cervical ripening.
We recently developed culture models of cyclic stretch to myometrium (Korita et al., 2002), fetal membrane (Terakawa et al., 2002) and uterine cervices (Yoshida et al., 2002) by using a computer-operated vacuum-driven cyclic stretch system, which mimics, at least partly, characteristic stimulation with distension of uterine cervices in parturition. In the present study, we used this culture model to investigate whether cyclic mechanical stretch can directly initiate chemotaxis of neutrophils and/or monocytes/macrophages in the process of cervical ripening.
Recently, the chemokine family has been characterized as a group of chemoattractants for various types of leukocytes (Rollins, 1997; Zlotnik and Yoshie, 2000; Patterson et al., 2002). Interleukin-8 (IL-8) and monocyte chemotactic protein-3 (MCP-3) are chemokines that induce chemotaxis of neutrophils and monocytes/macrophages respectively (Rollins, 1997; Zlotnik and Yoshie, 2000; Patterson et al., 2002; Menten et al., 2001). The objective of the present study was to investigate whether IL-8 and MCP-3 are expressed in the pregnant human uterine cervix and whether labour-like cyclic mechanical stretch augments IL-8 and MCP-3 production in cultured human uterine cervical fibroblast (CxF) cells.
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Materials and methods |
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Reagents
All reagents were purchased from Nacalai Tesque, Inc. (Japan), and were of analytical grade unless otherwise indicated.
Collection of cervical tissues and mucus
Uterine cervical tissues were obtained with informed consent from pre-menopausal non-pregnant women (n=5), first trimester pregnant women (n=2, 9 and 11 weeks), second trimester pregnant woman (n=1, 20 weeks) and term pregnant women (n=2, 37 and 38 weeks) at hysterectomy for gynaecological diseases (non-pregnant women for uterine myoma, four pregnant women for uterine cervical intraepithelial neoplasia, one pregnant woman for ovarian cancer). The tissues from two pregnant women at term were used to establish cultured human uterine cervical fibroblast (CxF) cells. The remaining tissues were snap-frozen using liquid nitrogen in blocks for mRNA extraction or embedded in OCT compound (Sakura Finetek Inc., USA) for immunohistochemical examination and stored at –80°C until used. The cervical mucus specimens were obtained with informed consent from term pregnant women after the onset of labour, without rupture of the membranes or clinical signs or symptoms of intrauterine infection (n=121). IL-8 and MCP-3 levels in the cervical mucus were measured using an enzyme-linked immunosorbent assay (ELISA) system specific to the respective substance (Biosource International Inc., USA). The study was approved by the ethics committee on human research at Kyoto University Graduate School of Medicine.
Preparation of cultured human uterine cervical fibroblast (CxF) cells
CxF cells were prepared by the explant method as previously reported (Yoshida et al., 2001, 2002). The cells from the tissues were grown to passage 6, and were used at that stage as cultured human uterine CxF cells in the further experiments in this study. An immunofluorescence study showed 99% positive staining for vimentin in CxF cells at the sixth passage, and <1% positive staining for both cytokeratin and -smooth muscle actin, indicating the high purity of the CxF cells, as previously described (Yoshida et al., 2001, 2002).
Immunohistochemical detection of IL-8 and MCP-3
Specimens were embedded in OCT compound (Sakura Finetek Inc., USA) and stored at –80°C. Sections 6 µm thick were incubated for 1 h at room temperature with anti-IL-8 mouse monoclonal antibody (1:20 dilution; Dako Japan Co., Japan) or anti-MCP-3 goat polyclonal antibody (1:40 dilution; Santa Cruz Biotechnology, USA). Normal mouse or normal goat serum (10 mg/ml; Dako Co., USA) was used as a negative control. Staining was detected using an avidin–biotin–peroxidase method kit for mouse monoclonal antibody or goat polyclonal antibody (Elite ABC; Vector Laboratories, USA) with 3,3'-diaminobenzidine as previously described (Itoh et al., 1998).
RT–PCR analysis of IL-8 and MCP-3
Total RNA was extracted from cervical tissue and cultured cervical fibroblast cells as previously described (Masuzaki et al., 1997). After reverse transcription of 2 µg of total RNA from human pregnant and non-pregnant uterine cervices and cultured human uterine cervical fibroblast cells using oligo(dT) primer (Promega, USA) and SuperscriptTM II (Gibco BRL, USA), the resulting single-stranded cDNA was subjected to PCR. Forward and reverse primers and programs used for amplifying portions of the cDNA of human IL-8 (Matsushima et al., 1988) and human MCP-3 (Opdenakker et al., 1994) were: IL-8 forward, 5'-AGATATTGCACGGGAGAA-3' and IL-8 reverse, 5'-GAAATAAAGGAGAAACCA-3'; MCP-3 forward, 5'-GCCTCTGCAGCACTTCTGTG-3' and MCP-3 reverse, 5'-CACTTCTGTGTGGGGTCAGC-3'. Forward and reverse primers for the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) coding region were purchased from Clontech Laboratories, Inc. (USA). The programs used for amplification of IL-8 and MCP-3 cDNA were: IL-8, 28 cycles of 94°C for 60 s, 55°C for 60 s, 72°C for 60 s; and MCP-3, 26 cycles of 94°C for 60 s, 58°C for 60 s, 72°C for 60 s. Cultured human promyelocytic leukaemia (HL-60) cells after 24 h of stimulation with 10 nmol/l 12-O-tetradecanoylphorbol 13-acetate and 10 ng/ml IL-1 were used as a positive control (Cavaillon, 1994).
Quantitative RT–PCR analysis of IL-8 and MCP-3
Quantitative RT–PCR of human IL-8, MCP-3 and GAPDH mRNA was carried out using real time TaqmanTM technology and the products were analysed using a Model 7700 sequence detector (Applied Biosystems, USA) (Yoshida et al., 2001). The forward and reverse primers, and Fam or Joe/Tamra probes used for targeting amplification of part of the human IL-8 (Matsushima et al., 1988), MCP-3 (Opdenakker et al., 1994) and GAPDH coding regions (Tso et al., 1985), were: IL-8 forward, 5'-CTCTTGGCAGCCTTCCTGATT-3'; IL-8 reverse, 5'-GGGTGGAAAGGTTTGGAGTATG-3'; MCP-3 forward, 5'-AAGCAGAGGCTGGAGAGCTACA-3'; MCP-3 reverse, 5'-AGTCCTGGACCCACTTCTGTGT-3'; GAPDH forward, 5'-GAAGGTGAAGGTCGGAGT-3'; GAPDH reverse, 5'-CTTCTACCACTACCC TAAAG-3'; IL-8 (Fam/Tamra) probe, 5'-Fam-TTAGCACTCCTTGGCAAAACTGCACC-Tamra-3'; MCP-3 (Fam/Tamra) probe, 5'-Fam-TGTCCCCGGGAAGCTGTAATCTTCAAGA-Tamra-3' and GAPDH (Joe/Tamra) probe, 5'-Joe-CCGACTCTTGCCCTTCGAAC-Tamra-3'. Cycling parameters used were 2 min at 50°C, 30 s at 60°C, and 5 min at 95°C, followed by 40 cycles of 20 s at 94°C and 1 min at 60°C. The human IL-8 or MCP-3 mRNA expression was estimated by dividing the IL-8 or MCP-3 threshold cycle (CT) values by GAPDH CT values as previously described (Yoshida et al., 2001).
Experimental protocol for cell culture and stretch experiment
CxF cells at the sixth passage were seeded in 6-well plates with flexible silicon bottoms (BIO Flex collagen-I; Flexcell International Co., USA). The culturing was conducted using minimum essential medium (MEM; Gibco BRL) containing 10% fetal bovine serum at 37°C in 95% air and 5% CO2 under humidified conditions. When the cells reached sub-confluency, the medium was replaced with fresh MEM without serum and the stimulation by cyclic mechanical stretch (repetition of 45 s stretch and 15 s release; –13 kPa, 15% elongation) was applied to the cells for 6–48 h using a computer-operated, vacuum-driven stretch device (Flexercell Strain Unit FX-3000; Flexcell International Co.) in the presence or absence of curcumin, an activator protein-1 (AP-1) inhibitor, as previously described (Korita et al., 2002; Yoshida et al., 2002). After 12, 24, 36 or 48 h of incubation, the media and cells were collected and stored at –20 and –80°C respectively. IL-8 and MCP-3 levels in the media were measured using an ELISA system specific to the respective substance (Biosource International Inc., USA). Expression of IL-8 and MCP-3 mRNA in the CxF cells was assessed by quantitative RT–PCR. After 48 h of incubation with cyclic mechanical stretch, the number of CxF cells was similar to that in the vehicle control without stretch. The human skin fibroblast cells, kindly supplied from New Drug Research Laboratories Ltd (Japan), were also subjected to the same stretch experiment for comparison.
Statistical analysis
Values were expressed as the means±SEM. Statistical significance was assessed by analysis of variance followed by Fisher's protected least significant difference test. P<0.05 was regarded as significant.
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Results |
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Expression of IL-8 and MCP-3 in non-pregnant and pregnant uterine cervical tissues and cultured CxF cells
RT–PCR analysis revealed IL-8 and MCP-3 mRNA expression in the non-pregnant uterine cervix, first, second and third trimester pregnant uterine cervices, cultured CxF cells and stimulated HL-60 cells (as a positive control) (Figure 1). The MCP-3 mRNA expression in CxF cells was more prominent than that in non-pregnant, first, second and third trimester pregnant uterine cervices (Figure 1).
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Immunohistochemically, strongly positive staining for IL-8 (Figure 2A) and MCP-3 (Figure 2C) were detected in both stromal cells and glandular cells in the pregnant uterine cervix at term, indicating that both IL-8 and MCP-3 were expressed widely in cervical tissues. Negative controls for both IL-8 and MCP-3 using normal mouse or goat IgG respectively, showed greatly reduced staining (Figures 2B, D). Stimulated HL-60 cells were used as a positive control (data not shown).
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Concentrations of IL-8 and MCP-3 in the cervical mucus obtained after the onset of labour
IL-8 concentrations in the cervical mucus specimens obtained from term pregnant women after the onset of labour with cervical canal dilatation of approximately 2, 3, 4, 5, 6 and 7 cm were 0.238±0.066 µg/ml (mean±SEM, n=25), 0.670±0.089 µg/ml (n=20), 0.802±0.210 µg/ml (n=13), 1.284±0.330 µg/ml (n=9), 0.623±0.189 µg/ml (n=5) and 0.595±0.125 µg/ml (n=7) respectively, which were significantly higher than those in specimens from women with closed cervical canals, 0.0692±0.020 µg/ml (n=19) (P<0.0001 for all) (Figure 3A).
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MCP-3 concentrations in the cervical mucus specimens obtained from term pregnant women after the onset of labour with cervical canal dilatation of approximately 2, 3, 4, 5, 6 and 7 cm were 5.31±0.096 mg/ml (n=25, P<0.0001), 5.87±1.20 mg/ml (n=20, P<0.0001), 5.15±1.48 mg/ml (n=13, P<0.0001), 6.04±5.76 mg/ml (n=9, P<0.05), 8.12±3.18 mg/ml (n=5, P<0.05) and 10.16±2.18 mg/ml (n=7, P<0.05), which were significantly higher than those in specimens from women with closed cervical canals, 3.60±0.17 mg/ml (n=19) (Figure 3B).
Effect of cyclic mechanical stretch on IL-8 and MCP-3 secretion from the cultured CxF cells
IL-8 concentrations in the medium of CxF cells after 12, 24, 36 and 48 h of incubation under stimulation with cyclic mechanical stretch were 6.9±0.3, 55±1.5, 161±12 and 161±10 pg/ml, which were significantly higher than those in the medium of cells without stimulation, 3.1±0.3, 7.2±0.4, 13.6±0.7 and 29±2.0 pg/ml respectively (n=4 and P<0.05 for all comparisons) (Figure 4A). On the other hand, the MCP-3 concentration in the medium of CxF cells after 12 h of incubation with cyclic mechanical stretch was not significantly different from that without stretch. However, MCP-3 concentrations in the medium of CxF cells after 24, 36 and 48 h of incubation under stimulation with cyclic mechanical stretch were 373±7.1, 780±34 and 1153±48 pg/ml, which were significantly higher than those without the stimulation, 232±6.8, 283±8.6 and 320±11 pg/ml respectively (n=4 and P<0.05 for all comparisons) (Figure 4C).
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Effect of cyclic mechanical stretch on IL-8 and MCP-3 secretion from the cultured human skin fibroblast cells
Cyclic mechanical stretch for 12–36 h significantly augmented IL-8 secretion from cultured human skin fibroblast cells; however, the magnitude of the increase (143–181%) was much lower than that in CxF cells (Figure 4B). By contrast, such augmentation was not observed for MCP-3 secretion in the same culture media of human skin fibroblast cells (Figure 4D).
Effect of cyclic mechanical stretch on IL-8 and MCP-3 mRNA expression in cultured CxF cells
IL-8 mRNA levels in the CxF cells after 6, 9, 12, 24, 36 and 48 h of incubation under stimulation with cyclic mechanical stretch were (in arbitrary units, AU; IL-8/GAPDH): 0.0023±0.00002, 0.0032±0.00004, 0.0096±0.0005, 0.0098±0.0017, 0.0120±0.00009 and 0.0056±0.0003, which were significantly higher than those without the stimulation: 0.0010±0.00001, 0.0016±0.00002, 0.0016±0.00029, 0.00062±0.00005, 0.0006±0.00019 and 0.0027±0.00057 respectively (n=6) (Figure 5A).
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The MCP-3 mRNA levels in the CxF cells after 6 and 9 h of incubation under stimulation with cyclic mechanical stretch, 0.0128±0.0009 and 0.0276±0.0029 AU were similar to those in CxF cells without the stimulation, 0.0184±0.0020 and 0.0263±0.0015 AU respectively (n=6) (Figure 5B). MCP-3 mRNA levels in the CxF cells after 12 h of incubation under cyclic mechanical stretch (0.058±0.002 AU (MCP-3/GAPDH) were slightly higher than those in CxF cells without stimulation (0.037±0.002 AU (MCP-3/GAPDH), but the difference was not statistically significant. However, the MCP-3 mRNA levels in the CxF cells after 24, 36 and 48 h of incubation under stimulation with cyclic mechanical stretch (MCP-3/GAPDH), 0.094±0.0082, 0.19±0.01 and 0.27±0.01 AU, were significantly higher than those in CxF cells without the stimulation, 0.060±0.004, 0.059±0.004 and 0.140±0.006 AU respectively (n=6 and P<0.01 for all) (Figure 5B).
Effect of curcumin on cyclic mechanical stretch-augmented IL-8 or MCP-3 secretion from cultured CxF cells
IL-8 concentrations in the culture medium of CxF cells after cyclic mechanical stretch for 30 h under conditions of co-treatment with 1, 5 and 15 µmol/l curcumin, started 1 h prior to stretch, were 23.9±0.7, 21.6±1.0 and 19.8±0.1 pg/ml respectively, which were significantly lower than that without curcumin co-treatment, 41.9±4.6 pg/ml (n=4, P<0.05 for all) (Figure 6A). By contrast, curcumin treatment did not affect the IL-8 concentration in the medium from CxF cells without cyclic mechanical stretch (Figure 6A).
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MCP-3 concentrations in the culture medium of CxF cells after cyclic mechanical stretch for 30 h under conditions of co-treatment with 1, 5 and 15 µmol/l curcumin, started 1 h prior to stretch, were 63.7±4.1, 51.3±3.8 and 43.7±1.7 pg/ml respectively, which were significantly lower than that without curcumin co-treatment, 91.6±13.1 pg/ml (n=6, P<0.05 for all) (Figure 6B). By contrast, curcumin treatment did not affect the MCP-3 concentration in the medium from CxF cells without cyclic mechanical stretch (Figure 6B).
Effect of curcumin on cyclic mechanical stretch-augmented IL-8 or MCP-3 mRNA expression in cultured CxF cells
The IL-8 mRNA level in cultured CxF cells after cyclic mechanical stretch for 30 h under conditions of co-treatment with 15 µmol/l curcumin was 0.00471±0.00021 AU (IL-8/GAPDH), which was significantly lower than that without curcumin co-treatment, 0.00758±0.00047 AU (n=4, P<0.05) (Figure 7A). By contrast, curcumin treatment did not affect the IL-8 mRNA level in cultured CxF cells without cyclic mechanical stretch (Figure 7A).
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The MCP-3 mRNA level in cultured CxF cells after cyclic mechanical stretch for 30 h under conditions of co-treatment with 15 µmol/l curcumin was 0.240±0.027 AU (MCP-3/GAPDH), which was significantly lower than that without curcumin co-treatment, 0.418±0.054 AU (n=4, P<0.05) (Figure 7B). By contrast, curcumin treatment did not affect MCP-3 mRNA expression in the cultured CxF cells without cyclic mechanical stretch (Figure 7B).
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Discussion |
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Marked infiltration of leukocytes occurs concomitantly with rapid degradation of extracellular matrix proteins during uterine cervical ripening (Junqueira et al., 1980
; Liggins, 1981; Kelly, 1994). Hegele-Hartung et al. (1989) reported that progesterone antagonist treatment augmented cervical ripening of pregnant guinea-pigs with concomitant infiltration of granulocytes and macrophages, demonstrating the possibility that progesterone withdrawal might induce the chemotaxis of both neutrophils and macrophages. In humans, however, a sharp decline of plasma progesterone levels occurs only after placental expulsion, and the contribution of progesterone withdrawal to the initiation of labour is still controversial. Our pilot study revealed that progesterone treatment elicits no significant alteration in cyclic mechanical stretch-induced augmentation of IL-8 nor MCP-3 secretion from CxF cells, although progesterone receptor mRNA expression was detected in CxF cells (M.Takemura, H.Itoh and N.Sagawa, unpublished findings). Thus, the possible involvement of progesterone in initiation of the chemotaxis of neutrophils and monocytes/macrophages in the process of cervical ripening in humans has not been fully elucidated.
Chemokines are chemoattractants for various types of leukocytes and have been classified into two major subfamilies, CXC and CC chemokines (Rollins, 1997; Zlotnik and Yoshie, 2000; Patterson et al., 2002). Most CXC chemokines predominantly attract neutrophils, whereas CC chemokines preferentially attract monocytes/macrophages and lymphocytes (Rollins, 1997; Zlotnik and Yoshie, 2000; Patterson et al., 2002).
Among the 15 currently known CXC chemokines, IL-8 was examined in the present study because it has been shown to act as a strong chemotactic factor for neutrophils at cervical ripening (Uchiyama et al., 1992; Calder, 1994; Chwalisz et al., 1994; Kelly, 1994; El Maradny et al., 1994; Maehara et al., 1996) and also that uterine cervical IL-8 production is markedly elevated at parturition (Sennstrom et al., 1997; Winkler et al., 1998, 1999b). Indeed, IL-8 was shown here to be widely expressed in the stroma of the human uterine cervix (Figure 2). Moreover, IL-8 concentrations in the cervical mucus specimens were elevated in parallel with 2–5 cm dilatation of the uterine cervical canal (Figure 3A), although IL-8 elevation in the cervical mucus was not direct evidence to support its increase in the cervical stromal tissues. Fibroblast cells are the major population of human uterine cervical stromal cells and are exposed to stimulation by cyclic mechanical distension by the presenting part of the fetus during labour. Based on these facts, we applied labour-like cyclic mechanical stretch to cultured human uterine CxF cells to test the hypothesis that mechanical stretch augments chemokine production in these cells. In the present study, labour-like cyclic mechanical stretch significantly augmented IL-8 secretion (Figure 4A) as well as mRNA expression (Figure 5A) in CxF cells, strongly suggesting that labour with cyclic contraction of the uterine corpus and descent of the presenting part of the fetus may mechanically distend the uterine cervix causing augmentation of IL-8 production in the uterine cervix. Such cyclic stretch-induced augmentation of IL-8 secretion was reported in endothelial cells (Okada et al., 1998) and alveolar epithelial cells (Vlahakis et al., 1999; Yamamoto et al., 2002). Maehara et al. (1996) revealed that continuous stretch increased IL-8 mRNA expression in the human fetal membrane.
It is interesting to speculate that cyclic mechanical stretch of the uterine cervix by labour may initiate chemotaxis of neutrophils via augmenting IL-8 production, and promotes degradation of uterine cervical collagen fibrils, finally contributing to the initiation and/or acceleration of cervical ripening.
In contrast to IL-8, which is one of the CXC chemokines, the involvement of CC chemokines in the process of cervical ripening has not been well understood to date. Currently, >27 CC chemokines have been identified, among which 
12 CC chemokines cause monocytes chemotaxis (Rollins, 1997; Zlotnik and Yoshie, 2000; Patterson et al., 2002). Amongst these 12 CC chemokines, our preliminary study showed that MCP-1 and regulated upon activation, normal T expressed and secreted (RANTES) mRNA were also expressed in the human uterine cervix, but that their concentrations in the medium of CxF cells were below the detection sensitivity of ELISA (15 pg/ml) (M.Takemura, H.Itoh, N.Sagawa unpublished findings). By contrast, MCP-3 protein was abundantly detected in both the uterine cervix and the culture medium of CxF cells (Figures 2C and 4C). Indeed, MCP-3 concentrations in the cervical mucus specimens were elevated in parallel with dilatation of the uterine cervical canal during labour (Figure 3B), although MCP-3 augmentation in the cervical mucus was not direct proof of its increase in the cervical stromal tissues. The present study further demonstrated that MCP-3 immunostaining was detected widely in the human uterine cervices from pregnant women (Figure 2C) and that labour-like cyclic mechanical stretch augmented MCP-3 secretion (Figure 4C) and mRNA expression (Figure 5B) in CxF cells, suggesting that cyclic contraction of the uterine corpus by labour may augment MCP-3 production in the uterine cervix through mechanical stretch of the cervix. Infiltrating monocytes/macrophages are hypothesized to be the main contributors in the production of pro-inflammatory cytokines such as IL-1, TNF- and IL-6 in inflammatory reactions (Cavaillon, 1994). It has been demonstrated that such pro-inflammatory cytokines play key roles in the process of cervical ripening by stimulating the production of various kinds of bioactive substances such as nitric oxide, prostaglandins and MMP, which accelerate the rapid degradation of extracellular matrix proteins in the uterine cervix (Kelly, 1994; Chwalisz and Garfield, 1998; Yoshida et al., 2001, 2002). Therefore, it is interesting to speculate that cyclic mechanical stretch during labour may augment MCP-3 production and initiate chemotaxis of monocytes/macrophages, which secrete pro-inflammatory cytokines and degrade uterine cervical collagen fibrils. As for the possible involvement of CC chemokines in the process of cervical ripening, Sugano et al. (2001) reported that platelet-activating factor (PAF) elevated both MCP-1 and RANTES mRNA expression in cultured human cervical fibroblast cells. Nevertheless, to our knowledge, the present study is the first report of the possible involvement of labour-like cyclic mechanical stretch as a physical factor acting in the augmentation of MCP-3 secretion in the complicated process of uterine cervical ripening.
We also examined the effects of cyclic mechanical stretch on IL-8 and MCP-3 secretion from human skin fibroblast cells for comparison. The present study demonstrated that cyclic mechanical stretch-induced augmentation of IL-8 secretion was much lower in human skin fibroblast cells (Figure 4B). Moreover, cyclic mechanical stretch did not alter MCP-3 secretion from cultured human skin fibroblast cells (Figure 4D), suggesting the interesting possibility that cyclic stretch-mediated up-regulation of MCP-3 production may be specific for uterine cervical fibroblast cells.
The AP-1 cascade is related to various types of immediate early genes (Du et al., 1995) and mediates stretch-associated signal transduction in several types of cells (Du et al., 1995; Wung et al., 1997; Park et al., 1999). Recently, we demonstrated that AP-1 also mediated cyclic stretch-associated augmentation of MMP-1 expression in CxF cells (Yoshida et al., 2002) and of prostacyclin synthase expression in cultured human myometrial cells (Korita et al., 2002). In the present study, curcumin, an inhibitor of AP-1 (Huang et al., 1991; Park et al., 1999; Korita et al., 2002; Yoshida et al., 2002), partially but significantly suppressed cyclic stretch-augmented MCP-3 as well as IL-8 secretion from CxF cells, suggesting the involvement of AP-1 in the cyclic stretch-associated signal transduction. Therefore, the findings of these in vitro studies suggest that cyclic distension of the uterine cervix by the presenting part of the fetus during labour may stimulate the AP-1 cascade and elevate both IL-8 and MCP-3 production in uterine CxF cells. However, the possible involvement of other signal transduction pathways must be considered in such cyclic stretch-associated augmentation of IL-8 and MCP-3 production, because the suppressive effect of curcumin was partial (Figures 6 and 7). Of course caution should be exercised in attributing the present in vitro findings to in vivo physiological phenomena caused by cyclic stretch in the complicated process of cervical ripening. Nevertheless the present in vitro data strongly suggest a possible involvement of cyclic mechanical stretch to chemotaxis of neutrophils as well as monocytes/macrophages in the process of cervical ripening. Since the present study did not show direct evidence that elevated IL-8 and MCP-3 cause local infiltration of neutrophils and monocytes/macrophages in the process of cervical ripening, further investigations will be needed to elucidate the involvement of cyclic stretch in the initiation and/or acceleration of cervical ripening.
In summary, both IL-8 and MCP-3 are produced by human cervical fibroblast cells in a manner that may be regulated at least partly by labour-like cyclic mechanical stretch. Based on these findings, we suggest that cyclic stretch during labour may play an important role in the initiation of leukocyte chemotaxis in the process of cervical ripening in humans.
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Acknowledgements |
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The authors thank Professor Kazuwa Nakao and Dr Masaki Harada, Department of Clinical Science and Medicine, Kyoto University Graduate School of Medicine, for cooperation in conducting the experiment with the Flexer Cell Strain Unit FX-3000. The authors also acknowledge Ms Akiko Kishimoto and Ms Akiko Abe for secretarial and technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports, Japan (No. 14657419, 14704042, 15390504, 15659393, 16390415), grants from the Smoking Research Foundation and the Kanzawa Medical Research Foundation.
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Notes |
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* Presented in part at the 50th Annual Meeting of the Society for Gynecologic Investigation.
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References |
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Submitted on March 26, 2004; resubmitted on May 6, 2004; accepted on May 14, 2004.
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