Human recombinant interleukin-1 alpha-mediated stimulation of procollagenase production and suppression of biosynthesis of tissue inhibitor of metalloproteinases in rabbit uterine cervical fibroblasts

Influence of human recombinant interleukin-1 (hrIL-1) on collagen metabolism was investigated with rabbit uterine cervical fibroblasts. Enzyme-linked immunosorbent assays for collagenase and tissue inhibitor of metalloproteinases (TIMP) indicated that hrIL-1 participates in both stimulation of procollagenase production and suppression of TIMP synthesis by uterine cervical cells. IL-1 did not modulate collagen synthesis. In addition, the sensitivity to IL-1 of uterine cervix from ovariectomized rabbits was augmented by estradiol-17 beta treatment. Thus it is proposed that IL-1 accelerates collagenolysis in the cervical tissue and its effect on uterine cervix is hormonally regulated.     

Dehydroepiandrosterone sulfate stimulates collagenase synthesis without affecting the rates of collagen and noncollagen protein syntheses by rabbit uterine cervical fibroblasts

The effects of dehydroepiandrosterone 3-sulfate (DHAS) and 17 beta-estradiol (E2) on collagen and noncollagen protein syntheses by rabbit uterine cervical cells were studied, and their effects on latent collagenase synthesis were compared. DHAS (1 X 10(-6) M) stimulated the synthesis of latent collagenase and did not affect the cell number and [3H]thymidine incorporation into DNA, whereas E2 had no effect on collagenase synthesis. On the other hand, neither DHAS (1 X 10(-6) M) nor E2 (1 X 10(-10)-1 X 10(-6) M) showed effects on collagen and noncollagen protein syntheses. These results suggest that the stimulative effect of DHAS on cervical ripening is mediated mainly by the stimulation of collagen catabolism, and that E2 does not concern the changes in the concentration of collagen and noncollagen protein in uterine cervix of the rabbit during pregnancy at term.     

Effects of prostaglandins on the production of collagenase by rabbit uterine cervical fibroblasts

Effects of prostaglandins on the production of collagenase by rabbit uterine cervical fibroblasts were investigated. Exogenous prostaglandin E2 (PGE2) and PGF2 alpha significantly stimulated the production of collagenase in a dose dependent manner, whereas PGI2 did not. Addition of arachidonic acid in the presence of absence of indomethacin to the cell culture did not show any increase in collagenase production. Recombinant human interleukin-1 (rhIL-1) also promoted the production of cervical collagenase independently of endogenous prostaglandin(s). Furthermore both exogenous PGE2 and PGF2 alpha enhanced the rhIL-1-induced collagenase production whereas PGI2 and/or indomethacin did not. These results suggested that exogenous PGE2 and PGF2 alpha but not endogenous prostaglandin(s) participate in cervical ripening and dilation by enhancing collagenase production by rabbit uterine cervical cells.     

Thrombospondin 2 deficiency in pregnant mice results in premature softening of the uterine cervix

The gradual disorganization of collagen fibers in the stromal connective tissue of the uterine cervix is characteristic of progressive cervical softening during pregnancy. A lack of thrombospondin (TSP) 2 has been shown to be associated with altered collagen fibril morphology of connective-tissue-rich organs such as skin and tendon. The goal of this study was to determine the role of TSP2 in cervical softening by studying a TSP2-null mouse line. Creep testing showed that, in the nonpregnant animal and on Day 10 of pregnancy, there was no difference between the cervical extensibility of the wild-type and the TSP2-deficient mice. However, by Day 14 of pregnancy, the TSP2-null mice showed 4.5-fold increase in cervical extensibility, and by Day 18, a 6.1-fold increase, when compared with wild-type mice. A further indicator of compromised cervical integrity was that, on Days 14 and 18 of pregnancy, the cervix of TSP2-null mice broke rapidly under standard loading conditions that did not break the cervix of wild-type mice. Western blotting showed that TSP2 was expressed in the cervix of mice on Days 14 and 18 of pregnancy but not on Day 10 or in the nonpregnant animal. As determined by immunohistochemistry, the amount of matrix metalloproteinase 2 (MMP2) in the cervix of TSP2-null mice increased 11-fold on Day 14 of pregnancy and 19-fold on Day 18. Thus, TSP2-null mice provide an animal model to assist in the understanding of the molecular basis of spontaneous, premature softening of the uterine cervix.     

Hormonal regulation of PGE2 and COX-2 production in rabbit uterine cervical fibroblasts.

Prostaglandins (PGs) cause uterine contraction to initiate labor at term. We investigated the effect of progesterone and 17beta-estradiol on the production of PGE2 in rabbit uterine cervical fibroblasts. When the cervical fibroblasts were treated with interleukin-1alpha (IL-1alpha), the level of PGE2 was augmented in a time- and dose-dependent manner. The IL-1alpha-augmented PGE2 level was almost completely suppressed by progesterone and 17beta-estradiol at the physiological concentration (0.01 microM), whereas a slight decrease in the basal level of PGE2 was observed in the cervical fibroblasts treated with both hormones at a pharmacological concentration (1 microM). In addition, the level of PGE2 augmented by IL-1alpha was due to the increase of cyclooxygenase (COX) activity, which was inhibited by progesterone and 17beta-estradiol as well as by indomethacin and a specific COX-2 inhibitor, NS-398, but not by the well-known COX-1 inhibitor, aspirin. Furthermore, progesterone and 17beta-estradiol suppressed the IL-1alpha-augmented COX-2 production but not the constitutive production of COX-1 in rabbit uterine cervical fibroblasts. These results suggest that progesterone and 17beta-estradiol prevent the initiation of labor by inhibiting PGE2 production after the suppression of COX-2 production during pregnancy in the rabbit.     

Effects of RU486 on the interstitial collagenase in the process of cervical ripening in the pregnant rat.

Changes in interstitial collagenase activity in the rat uterine cervix during ripening were clarified in a time-dependent manner. Premature delivery was induced by an antiprogesterone agent, RU486, for rats in late pregnancy. The presence of interstitial collagenase in the extract from the rat cervical tissue was demonstrated, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using the natural and unaffected collagen as a substrate. The collagenase activity was determined as the release of digested peptides from the radio-labeled collagen. Our experiments with RU486 were performed in rats on the 18th day of pregnancy. A single administration of RU486 (15 mg/kg) resulted in the premature delivery of all treated rats within 30 h after the injection (average time was 23.9 h). The marked increase in cervical wet weight was observed up to the time to premature delivery along with a significant acceleration from 18 h after the administration of RU486. In this state, the cervical collagenase activity was enhanced, the highest levels being recorded at 21 h after the administration. The interstitial collagenase in the uterine cervix appears to play a significant role in the regulation mechanisms of cervical ripening in late pregnant rats.     

Effect of dehydroepiandrosterone sulfate on collagenase production in rabbit uterine cervix culture

Rabbit uterine cervical explants were found to produce a typical collagenase, latent form, in tissue culture, 4-Aminophenylmercuric acetate and trypsin were potent activators of the enzyme. The enzyme was purified simply in one step of CM-52 cellulose ion-exchange chromatography, and then further characterized. Addition of dehydroepiandrosterone sulfate (DHAS) to culture medium significantly stimulated collagenase production, but DHAS did not directly activate the enzyme. In addition, dehydroepiandrosterone and 17 beta-estradiol, the main metabolites of DHAS in vivo, depressed enzyme production. Our previous result, that increases in cytoplasmic DHAS-binding protein in rabbit uterine cervices parallel the progress of pregnancy, and these results suggest that DHAS might have direct actions toward cervical ripening.     

Biochemical evidence of collagenase-mediated collagenolysis as a mechanism of cervical dilatation at parturition in the guinea pig.

Dilatation of the uterine cervix at parturition is accompanied by a remarkable alteration in its biomechanical characteristics leading to a significant reduction in its strength and stiffness. Our previous studies and those of others have suggested the involvement of collagenolysis leading to cervical dilatation. This study provides further evidence for the occurrence of collagenolysis in the dilated guinea pig cervix at birth. The changes in collagenase and collagenase inhibitory activity in vivo in cervices of nonpregnant, pregnant, and postpartum guinea pigs were determined. There were no significant changes in procollagenase, collagenase inhibitory activity, and net procollagenase in animals at 25 and 50 days of gestation compared with nonpregnant animals. At parturition (68 +/- 2 days), there was a 6-fold increase in procollagenase, a 26-fold increase in collagenase inhibitory activity, and a 2-fold increase in net procollagenase activity. Cervices in organ culture obtained at birth produced 2.9-fold more procollagenase, 1.6-fold more collagenase inhibitory activity, and a 10-fold increase in net procollagenase activity when compared to nonpregnant or 25-day pregnant animals. These studies indicate that dilatation of the guinea pig cervix at parturition involves collagenase-mediated degradation of collagen in the cervix.     

Effect of dehydroepiandrosterone sulfate on uterine cervical ripening in late pregnancy

In order to elucidate the effects of dehydroepiandrosterone sulfate (DHAS) on softening and dilatation of the uterine cervix, changes of oestriol, 17 beta-oestradiol and progesterone levels in serum and cervix, Bishop score and collagenase activity in the cervical tissue were assessed in pregnant women before and after treatment with DHAS. 17 beta-oestradiol level in the serum and cervical tissue markedly increased after the administration of DHAS, while oestriol level remained unchanged. Serum progesterone level did not change in the majority of cases, while it decreased within several hours in patients in whom delivery was accomplished within 24 hours after the administration of DHAS. Among the factors connected with the Bishop score, effacement and consistency of the cervix were remarkably improved by DHAS administration. Total collagenase activity in the cervical tissue of patients treated with DHAS was elevated by an average of 152%. These results suggest that DHAS is potent in ripening the uterine cervix through an activation of collagenase activity induced by the enhanced conversion to 17 beta-oestradiol. Thus, DHAS administration in the late stage of pregnancy is valuable in prepartal treatment for induction of labour.     

Calmodulin regulates the interleukin 1-induced procollagenase production in human uterine cervical fibroblasts

Interleukin 1 (IL-1) stimulates the synthesis of collagenase in human uterine cervical fibroblasts. This inductive effect of IL-1 on collagenase production was augmented by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific inhibitor of calmodulin, in a dose-dependent manner. The apparent collagenase activity observed in the culture medium of the cells treated with IL-1 and 40 microM W-7 was about three times higher than that produced by the cells treated with IL-1 alone. The immunoblotting with the specific antibody against human collagenase showed that the increased collagenase activity resulted from the accelerated biosynthesis of collagenase. Another calmodulin inhibitor, trifluoperazine, enhanced the effect of IL-1 on collagenase production similarly. However, the effect of N-(6-aminohexyl)-1-naphthalenesulfonamide, the weakest inhibitor of calmodulin, was negligible. These results suggest that W-7 enhances the collagenase production by specifically inhibiting calmodulin and that calmodulin may act as a suppressor of the IL-1-induced collagenase production in human uterine cervical fibroblasts.     

Characterization of fibroblastic cell plasticity in the lamina propria of the rat uterine cervix at term

Different organs contain fibroblasts with specific features and functions, indicating the complexity of fibroblast biology. In the rat cervical stroma, fibroblasts are preferentially located in the fibrous ring that surrounds the mucous layer. The purpose of this study was to investigate the morphological features and immunophenotype of fibroblastic cells of the uterine cervix in cycling, pregnant, and postpartum rats. Expression of the cytoskeletal proteins desmin, vimentin, and alpha-smooth muscle actin (alpha-SMA) were studied by immunohistochemistry. The optical density of immunohistochemical staining was quantified by image analysis. The ultrastructural features of fibroblastic cells were observed under transmission electron microscopy. Cervical fibroblastic cells always expressed vimentin and desmin but never alpha-SMA. During the first half of pregnancy (Day 5 [D5] to D14), desmin intensity values were similar to those of cycling and postpartum fibroblasts. In contrast, a strong expression of desmin was found from D15 to D22, with maximal expression at term (D23). Immunohistochemical expression for vimentin was constant throughout pregnancy and showed no differences with cycling and postpartum uterine cervices. Stromal cells from cycling and early pregnant rats displayed ultrastructural features characteristic of typical fibroblasts. In contrast, at the end of pregnancy, fibroblasts differentiated and showed increased secretory characteristics, reaching the ultrastructural features of a myofibroblast. Based on the differential expression of desmin and the electron microscopic observations, the foregoing results showed a modulation of the fibroblastic phenotype in the uterine cervix during pregnancy. To our knowledge, this is the first report that addresses the presence of myofibroblasts derived from resident fibroblasts in the fibrous ring of the rat uterine cervix. Fibroblastic-myofibroblastic cell plasticity may have implications in the physiological changes displayed in the uterine cervix during pregnancy, parturition, and postpartum involution.     

Sensory nerves and neuropeptides in uterine cervical ripening

At the time of parturition (fetal delivery) the uterine cervix must "ripen," becoming soft, pliable, and dilated to accommodate the fetus' delivery. The fundamental processes underlying cervical ripening remain poorly understood. Knowledge that abundant autonomic and sensory nerves supply the uterine cervix, that transection of afferent nerves supplying the cervix blocks parturition, and that some of the changes in the cervix resemble those seen in inflammatory reactions suggests nerves may have a role in the cervical ripening changes. The present study utilized immunohistochemistry, plasma extravasation, and solution hybridization-nuclease protection assay to elucidate the complement of primary afferent nerves and some receptors in the rat cervix during pregnancy, and to determine if they may have roles in the ripening process at term. This study revealed an abundance of nerves associated with the cervical vasculature and myometrial smooth muscle containing immunoreactivity for substance P, calcitonin gene-related peptide, secretoneurin, and nitric oxide synthase throughout pregnancy. Many of these are small unmyelinated capsaicin-sensitive C-fibers. Substance P- (NK1-) and calcitonin gene-related peptide receptors were apparent on uterine cervix vasculature from pregnant, parturient, and postpartum rats. NK1 receptor mRNA was maximal at 20 days of pregnancy. Plasma extravasation of i.v. administered Evans Blue or Monastral Blue was most pronounced at parturition (shortly after NK1 mRNA is maximal); this was similar to plasma extravasation evoked by i.v. administration of substance P or capsaicin-treatment. This study revealed new data about the nervous system of the rat uterine cervix and that these nerves and their transmitters could very well be part of a neurogenic inflammatory process involved in cervical ripening.     

Macrophage trafficking in the uterus and cervix precedes parturition in the mouse.

Immune activation is implicated in the etiology of preterm labor, but little is known about macrophage number or distribution in the uterus or cervix at term. This study tested the hypothesis that macrophages migrate into the reproductive tract before the onset of parturition. Paraffin-embedded sections from the mid-uterine horn and cervix of C3/HeN mice on Days 15 and 18 of pregnancy, the day of birth (Day 19), and 1 day postpartum were stained with a pan-macrophage marker to analyze cell numbers and distribution. During pregnancy, uterine macrophages were dispersed in endometrium, usually associated with vasculature and subluminal epithelium. In myometrium, macrophages were clustered in stromal connective tissue; near term and postpartum, cells appeared to surround the muscle bundles. Total macrophage numbers were increased on Day 15 relative to those in nonpregnant controls, declined before birth, and increased postpartum. In the cervix, macrophages congregated in subepithelium, often perivascular or near ganglia. Macrophage numbers in the cervix peaked on Day 18, then declined to nonpregnant levels by the day after birth. Thus, macrophage numbers in the uterus were inversely related to those in the cervix. These findings raise the possibility that macrophages and their products may be involved in uterine contractility and cervical remodeling during the processes of parturition.     

Regulation of hyaluronan synthases in mouse uterine cervix

We examined the expression pattern of hyaluronan synthase (HAS) mRNAs in the uterine cervix of pregnant mice. The expression levels of HAS-1 and -2 mRNAs peaked at delivery, whereas that of HAS-3 mRNA peaked on the 15th day of pregnancy. The regulation of HAS mRNA expression was examined in pregnant mouse uterine cervical fibroblasts. The expression of HAS-1, -2, and -3 mRNAs was significantly augmented by interleukin-1beta (IL-1beta). Progesterone significantly interfered with expression of HAS-1 and -2 mRNAs, but significantly increased the expression of HAS-3 mRNA. Low-molecular-weight hyaluronan significantly enhanced only the expression of HAS-1 mRNA. These results indicate that HAS in the uterine cervix is regulated in a complex manner by IL-1beta, progesterone, and low-molecular-weight hyaluronan, of which changes in the cervical tissue and serum closely participate in uterine cervical ripening and/or inflammation.     

Uterine epithelial cells synthesize granulocyte-macrophage colony-stimulating factor and interleukin-6 in pregnant and nonpregnant mice.

Cytokine secretion by endometrial cells from estrous and mated mice was measured using specific bioassays. The granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) contents of uterine intraluminal fluid were elevated greater than 20-fold and 250-fold respectively following mating, and both cytokines were synthesized in abundance in vitro by uterine cells harvested at estrus and on Day 1 of pregnancy. Synthesis was not impaired in genetically lymphocyte-deficient nude, SCID, or beige mice. To determine the cellular origin of the cytokines, a panning technique employing monoclonal antibodies against a range of leukocyte and other lineage markers was used to isolate uterine cell subsets in vitro. These experiments identified glandular and/or luminal epithelial cells as the major source of GM-CSF and IL-6 in estrous and pregnant uteri. Stromal fibroblasts also synthesized IL-6, as did macrophages in mated mice. Epithelial cells harvested from midgestation uteri secreted GM-CSF and IL-6 in quantities similar to those of cells from estrous and mated mice. Bioactivities of both cytokines derived from epithelial cells were neutralized by specific antibodies, and size-exclusion chromatography of conditioned media from uterine cells revealed peaks of GM-CSF and IL-6 bioactivity with M(r) 23,000 and 23,000-26,000, respectively. Bioassay of luminal fluids and culture supernatants were negative for the cytokines interleukin-1, interleukin-2, interleukin-3, and tumor necrosis factor-alpha. These studies identify murine uterine epithelium as a potent source of the cytokines GM-CSF and IL-6, which we postulate have potentially important functions in pregnancy through actions on target cells in both the uterus and the conceptus.     

Calmodulin differentially modulates the interleukin 1-induced biosynthesis of tissue inhibitor of metalloproteinases and matrix metalloproteinases in human uterine cervical fibroblasts.

The effects of a specific calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on the synthesis of tissue inhibitor of metalloproteinases (TIMP) and precursor of matrix metalloproteinase 1/tissue collagenase (proMMP-1) and matrix metalloproteinase 3/stromelysin (proMMP-3), were examined using human uterine cervical fibroblasts in culture. When the cells were treated with human recombinant interleukin 1 alpha, the synthesis of TIMP, proMMP-1, and proMMP-3 was greatly enhanced along with the increase in the steady-state levels of mRNAs for respective proteins. The treatment of the cells with human recombinant interleukin 1 alpha and W-7 further augmented the production of proMMPs-1 and -3 and the accumulation of their mRNAs. In contrast, TIMP production and its steady-state mRNA level were reduced considerably under these conditions. Similar observations were made with another calmodulin inhibitor, trifluoperazine, but not with N-(6-aminohexyl)-1-naphthalenesulfonamide, the weakest inhibitor for calmodulin. These results indicate that calmodulin is required for the interleukin 1-enhanced synthesis of TIMP but it is a suppressor for the synthesis of proMMPs-1 and -3.     

Immunolocalization of proinflammatory cytokines in myometrium, cervix, and fetal membranes during human parturition at term.

Each of the proinflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF) alpha has been identified in reproductive tissues during labor. The cellular origin of these cytokines is unclear. The aim of this study was to localize these proinflammatory cytokines in myometrium (upper and lower segment), cervix, and fetal membranes at term. Biopsies were taken from women undergoing cesarean section either before or after the onset of labor. Immunohistochemistry was used to localize each of the cytokines IL-1beta, IL-6, IL-8, and TNFalpha. Leukocytes were localized using an antibody to CD45. In myometrium and cervix, immunostaining for IL-1beta was predominantly in leukocytes. In fetal membranes, IL-1beta localized to leukocytes and to the stromal cells of the decidua. In myometrium, IL-6, IL-8, and TNFalpha were restricted to leukocytes, which were present in greater numbers in tissue obtained during labor. In cervix, IL-6, IL-8, and TNFalpha localized to leukocytes and glandular and surface epithelium. IL-8 also localized to cervical stromal cells. In fetal membranes, IL-6 and TNFalpha were expressed by decidual stromal cells, infiltrating leukocytes, and extravillous trophoblasts. In membranes, IL-8 localized to leukocytes in the chorion but was not detected in the amnion. In fetal membranes collected at labor, IL-8 was expressed in decidual stromal cells. Infiltrating leukocytes are a major source of cytokines in uterine tissues during labor.