Regulation of matrix metalloproteinase expression by estrogen in fibroblasts that are derived from the pelvic floor

OBJECTIVE: The purpose of this study was to determine whether estrogen suppresses matrix metalloproteinase-2 and -9 proenzyme expression by fibroblasts that are derived from the supportive connective tissue of the pelvic floor. STUDY DESIGN: A primary fibroblast culture that was developed from a biopsy specimen of the arcus tendineus was treated with interleukin-1 beta (10-15 ng/mL), transforming growth factor-beta 1 (5-15 ng/mL), 17 beta-estradiol (10(-9)-10(-7) mol/L), and Imperial Chemical Industries (ICI) 182 780 (10(-8)-10(-6) mol/L). Cellular and extracellular protein were analyzed by Western blotting and substrate zymography, respectively, for the effect of each treatment on the amount of pro-matrix metalloproteinase-2 and -9 and the membrane type 1 matrix metalloproteinase protein. RESULTS: Both cellular and extracellular pro-matrix metalloproteinase-2 protein were increased by transforming growth factor-beta1 (P =.01) and decreased by estradiol (P <.001) and ICI 182 780 (P =.02 and.002, respectively). Membrane type 1 matrix metalloproteinase was not affected by estradiol, ICI 182 780, interleukin-1 beta, or transforming growth factor-beta 1. Extracellular pro-matrix metalloproteinase-9 was increased by the cytokines interleukin-1 beta (P <.001) and transforming growth factor-beta1 (P <.001) and decreased by estradiol (P <.001) and ICI 182 780 (P <.001). CONCLUSION: The proenzymes of the tissue-degrading matrix metalloproteinases -2 and -9 are decreased by 17-beta estradiol and ICI 182 780.     

Quantitation of tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 in the effusions of ovarian epithelial neoplasms.

OBJECTIVE: Our objective was to determine the presence and quantities of multifunctional cytokines in cyst and ascites fluids obtained from patients with ovarian cancer. STUDY DESIGN: Cyst and ascites fluids obtained from 35 patients with ovarian epithelial neoplasms were analyzed for the multifunctional cytokines tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. The fluids were classified on the basis of the pathologic diagnosis of the tumor tissue. Data were evaluated by analysis of variance. RESULTS: The fluids from patients with papillary forms of adenocarcinoma had high levels of all three cytokines when compared with all other pathologic groups. Interleukin-6 was significantly higher than tumor necrosis factor-alpha or interleukin-1 beta in fluids from all diagnostic categories (p < 0.01). Interleukin-6 levels were significantly higher in fluids from patients with papillary serous cystadenocarcinoma (817 +/- 137 pg/ml) and papillary adenocarcinoma (838 +/- 171 pg/ml) (p < 0.01). Interleukin-1 beta was significantly elevated (p < 0.01) in neoplastic effusions from papillary serous cystadenocarcinomas (53 +/- 8 pg/ml), mucinous cystadenomas (51 +/- 12 pg/ml), and endometrioid carcinomas (54 +/- 18 pg/ml). Tumor necrosis factor-alpha was highest in fluids from patients with papillary adenocarcinoma (46.2 +/- 22.8 pg/ml); however, these levels were not significantly different from the mean quantities of tumor necrosis factor-alpha in other fluids. CONCLUSIONS: The detection of interleukin-1 beta and interleukin-6 in neoplastic effusions provides possible evidence for a host immune response to ovarian cancer. These multifunctional cytokines have been implicated in growth stimulation and cytotoxicity of ovarian tumor cells.     

Identification of matrix metalloproteinase-9 in amniotic fluid and amniochorion in spontaneous labor and after experimental intrauterine infection or interleukin-1 beta infusion in pregnant rhesus monkeys.

OBJECTIVE: The purpose of this study was to examine the roles of intrauterine infection, inflammation, and spontaneous labor on the expression of matrix metalloproteinase-9 in fetal membranes and amniotic fluid of late pregnant rhesus monkeys. STUDY DESIGN: Pregnant rhesus monkeys with timed gestations were chronically catheterized to allow serial sampling of amniotic fluid before and during experimentally induced intrauterine inflammation. Six animals received group B streptococci into the chorionic-decidual space, and 4 animals received intra-amniotic interleukin-1 beta infusions (10 microg). Three additional animals were serially sampled by amniocentesis through late pregnancy until spontaneous term labor. Amniotic fluid samples were examined by zymography for matrix metalloproteinase-9 and -2 and Western immunoblot for matrix metalloproteinase-9 and -2 and tissue inhibitors of metalloproteinase-1 and -2. Fetal membranes were obtained at cesarean delivery during labor (before rupture), formalin fixed, and embedded in paraffin for immunocytochemistry of matrix metalloproteinase-9 and in situ hybridization of matrix metalloproteinase-9 messenger RNA. Tissues from 2 additional animals were collected as age-matched non-labor controls. RESULTS: In amniotic fluid, the 92-kd latent matrix metalloproteinase-9 was detectable in late pregnancy and increased dramatically, followed by the appearance of the 83-kd active form before spontaneous term delivery. Amniotic fluid matrix metalloproteinase-2 levels (both latent and active forms) remained relatively constant throughout pregnancy and in labor. Both bacteria and interleukin-1 beta rapidly increased the signal of latent matrix metalloproteinase-9 without a consistent increase in the active form before the onset of labor. Chorionic-decidual inoculation of group B streptococci was followed by the expression of latent matrix metalloproteinase-9 before the appearance of group B streptococci in amniotic fluid or the onset of labor. Matrix metalloproteinase-2 increased to a new steady-state level or remained unchanged after group B streptococci inoculation or interleukin-1 beta infusion, respectively. Amniotic fluid tissue inhibitors of metalloproteinase-1 declined and tissue inhibitors of metalloproteinase-2 remained unchanged during early group B streptococci infection, after interleukin-1 beta infusion and on the day of spontaneous term labor. However, both tissue inhibitors of metalloproteinase-1 and -2 levels increased after preterm labor that was induced by group B streptococci. Immunocytochemistry localized matrix metalloproteinase-9 protein to amnion and chorion epithelial and mesenchymal cells and decidual stromal cells. Granular matrix metalloproteinase-9 staining was observed in the connective tissue layer of inflamed fetal membranes. In situ hybridization for messenger RNA confirmed the production of matrix metalloproteinase-9 by amnion and chorion. CONCLUSION: Bacterial- and interleukin-1 beta-induced preterm labor and spontaneous term labor are preceded and accompanied by progressive increases in amniotic fluid matrix metalloproteinase-9 (92 kd) in rhesus monkeys. Amniotic fluid matrix metalloproteinase-9 may serve as a clinical marker for the onset of both preterm and term labor.     

Serum and follicular fluid cytokines in polycystic ovary syndrome during stimulated cycles

OBJECTIVE: To investigate the serum and intrafollicular tumor necrosis factor-alpha and interleukin-6 concentrations in infertile women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF). METHODS: Thirty-one patients with PCOS undergoing IVF were studied. Thirty-nine normally ovulating women matched for age and body mass index and undergoing IVF for male infertility were the control group. Serum tumor necrosis factor-alpha, interleukin-6, and estradiol levels were assayed before recombinant follicle-stimulating hormone stimulation under gonadotropin-releasing hormone analogue suppression and 34-36 hours after human chorionic gonadotropin (hCG) administration at the time of the oocyte retrieval. Cytokine and estradiol concentrations were also evaluated in the follicular fluids obtained at the time of oocyte retrieval. RESULTS: The patients with PCOS had higher serum and follicular fluid tumor necrosis factor-alpha and interleukin-6 concentrations (P <.001) and lower follicular fluid estradiol levels (P <.05) than control women. In both groups, the serum tumor necrosis factor-alpha, interleukin-6, and estradiol values increased significantly after hCG stimulation. In both groups, the follicular fluid cytokine concentrations were higher than those found in the serum. In the PCOS women the follicular fluid tumor necrosis factor-alpha values were significantly and inversely correlated to the follicular fluid estradiol values (rho = -0.79; P <.001); this correlation was not found in the control subjects. CONCLUSION: In infertile women with PCOS, 1). serum and follicular fluid interleukin-6 and tumor necrosis factor-alpha values were higher than those found in control women, 2). the cytokine concentrations were higher in the follicular fluid than in the serum, and 3). the intrafollicular tumor necrosis factor-alpha concentrations were significantly and inversely correlated to the estradiol levels. These results suggest an involvement of the immune system in PCOS.     

Endotoxin-induced cytokine production of monocytes of third-trimester pregnant women compared with women in the follicular phase of the menstrual cycle

OBJECTIVE: Little is known about the function of the innate immune response during pregnancy. We therefore investigated monocyte cytokine production, as a measure of monocyte function, in pregnant women compared with nonpregnant women. STUDY DESIGN: Whole blood of women in the follicular phase (day 5-6) and of healthy pregnant women (30 weeks) was collected and stimulated with endotoxin (2 microg/mL). After incubation for 4 hours (37 degrees C, 5% carbon dioxide), red blood cells were lysed and white blood cells were permeabilized, followed by staining with anti-CD14 (fluorescein isothiocyanate labeled) and with phycoerythrin-labeled tumor necrosis factor-alpha, interleukin-1beta, or interleukin-12. The cells were analyzed by flow cytometry after fixation. Results are expressed as a percentage cytokine producing cells after endotoxin stimulation. Statistical analysis was performed with the Mann-Whitney U test (P <.05). RESULTS: Compared with the percentage endotoxin-induced cytokine producing peripheral monocytes in women in the follicular phase, this percentage in pregnancy was decreased for interleukin-12 (mean 6.63 +/- 1.34 vs 3.34 +/- 0.87, P <.05) and tumor necrosis factor-alpha (mean 50.20 +/- 5.80 vs 31.29 +/- 5.57, P >.05). No significant difference was seen in the production of interleukin-1beta (mean 58.22 +/- 11.09 vs 47.18 +/- 7.88, P >.05). CONCLUSION: The percentage of interleukin-12 and tumor necrosis factor-alpha producing monocytes is decreased in pregnant women compared with nonpregnant women, suggesting that pregnancy is a proinflammatory state.     

Amniotic fluid tumor necrosis factor-alpha and interleukin-1 in a rabbit model of bacterially induced preterm pregnancy loss.

OBJECTIVE: The objective of this study was to determine whether the cytokines tumor necrosis factor-alpha, interleukin-1 alpha, and interleukin-1 beta were produced in the amniotic fluid of the rabbit after intracervical inoculation with Escherichia coli. STUDY DESIGN: Timed pregnant rabbits on day 21 (70% of gestation) were inoculated with a hysteroscope intracervically with 10(4) to 10(5) colony-forming units Escherichia coli or sterile saline solution. Escherichia coli-inoculated animals (N = 16) were put to death at 4, 8, 12, and 16 hours after inoculation. Control animals (N = 6) were put to death at similar intervals. At death, cultures were taken from endometrium, amniotic fluid, peritoneum, and blood. Amniotic fluid was collected and assayed for tumor necrosis factor bioactivity by a modified fibroblast cytotoxic assay in L929 cells, for interleukin-1 alpha, and interleukin-1 beta with a specific radioimmunoassay, and for prostaglandin E2 and prostaglandin F2 alpha by radioimmunoassay. RESULTS: Levels of amniotic fluid tumor necrosis factor-alpha, interleukin-1 alpha, and interleukin-1 beta were elevated as early as 4 hours after inoculation in some animals and by 12 to 16 hours after inoculation in all. Levels of all three cytokines correlated significantly with time from intracervical inoculation with Escherichia coli (p < 0.05). Levels of amniotic fluid prostaglandin E2 and prostaglandin F2 alpha correlated significantly with time from intracervical inoculation with Escherichia coli (p < 0.05). CONCLUSIONS: Levels of tumor necrosis factor-alpha, interleukin-1 alpha, interleukin-1 beta, prostaglandin E2 and prostaglandin F2 alpha are elevated in the amniotic fluid of rabbits after intracervical inoculation with Escherichia coli. Similarity exists between elevations of amniotic fluid cytokines in this model and in cases of intraamniotic infection and preterm labor unresponsive to tocolytics in humans. Modulation of cytokines may offer a strategy for improvement of outcome in this experimental model of infection-induced pregnancy loss.     

Elastin metabolism in pelvic tissues: is it modulated by reproductive hormones?

OBJECTIVE: The purpose of this study was to investigate the effect of relaxin on extracellular matrix protein expression in pelvic fibroblasts that were cultured from women with stress urinary incontinence compared with asymptomatic control subjects. STUDY DESIGN: Periurethral vaginal wall fibroblasts from premenopausal women with stress urinary incontinence and continent women (in both the proliferative and secretory phase of the menstrual cycle) were stimulated with increasing concentrations of relaxin (0-500 ng/mL). The supernatant was sampled for matrix metalloproteinase-2 and -9 by zymography. Tissue inhibitors of metalloproteinase-1 and -2 and alpha-1 antitrypsin were evaluated with Western blot. Total elastase activity was measured by generation of free amino groups from succinylated elastin. Increasing concentrations of alpha-1 antitrypsin were added to cell lysate to evaluate total elastase activity inhibition. RESULTS: Proliferative-phase stress urinary incontinence fibroblasts demonstrated an increase in matrix metalloproteinase-2 and no change in matrix metalloproteinase-9 and tissue inhibitors of metalloproteinase-1 and -2 expressions with increasing relaxin concentrations. Cells from control subjects showed increased expression of matrix metalloproteinase-2 and -9, but no change in tissue inhibitors of metalloproteinases. Secretory-phase stress urinary incontinence fibroblasts showed no response in matrix metalloproteinase or tissue inhibitors of metalloproteinase expressions with relaxin stimulation. Secretory-phase control fibroblasts reacted by increasing matrix metalloproteinase-2 and -9 and tissue inhibitors of metalloproteinase-2. With respect to total elastase activity and alpha-1 antitrypsin expression, increasing doses of relaxin appear to increase elastolytic activity in stress urinary incontinence cells by decreasing the expression of alpha-1 antitrypsin in proliferative phase cells or increasing the total elastase activity in secretory phase cells. Fibroblast total elastase activity was inhibited by increasing concentrations of alpha-1 antitrypsin. CONCLUSION: Elastase activity appears to be increased in relaxin-stimulated stress urinary incontinence fibroblasts by either decreased inhibitor (alpha-1 antitrypsin) production or increased elastase activity.     

Dexamethasone or interleukin-10 blocks interleukin-1beta-induced uterine contractions in pregnant rhesus monkeys

OBJECTIVE: The purpose of this study was to determine whether treatment with the immune modulators dexamethasone or interleukin-10 prevents interleukin-1beta-induced uterine contractions in a nonhuman primate model. STUDY DESIGN: Thirteen chronically instrumented rhesus monkeys at 135 +/- 1 days of gestation (term, 167 days) received one of three interventions: (1) intra-amniotic interleukin-1beta (10 microg) infusion with maternal dexamethasone (1 mg/kg) intravenously every 6 hours for 1 day before interleukin-1beta and for 2 days thereafter (n = 4), (2) intra-amniotic interleukin-1beta infusion with maternal interleukin-10 (25 microg/kg) given intravenously and 100 microg interleukin-10 given intra-amniotically before the interleukin-1beta and continued every 8 hours for 3 days (n = 5), and (3) intra-amniotic interleukin-1beta administered alone (n = 5). Uterine activity was monitored continuously and quantified as the hourly contraction area (millimeters of mercury times seconds per hour) in all groups until delivery. Amniotic fluid was sampled for leukocyte counts and assayed for prostaglandins E(2) and F(2)alpha, cytokines interleukin-1beta, interleukin-6, interleukin-8, tumor necrosis factor-alpha, interleukin-10, and interleukin-1 receptor antagonist by specific assays. Maternal and fetal blood were assayed for cortisol, dehydroepiandrosterone sulfate, and estradiol. RESULTS: Interleukin-1beta infusion in the absence of immune modulators resulted in an increase in uterine activity and amniotic fluid proinflammatory cytokines, prostaglandins, and leukocytes. Dexamethasone and interleukin-10 treatment significantly reduced interleukin-1beta-induced uterine contractility (P <.05) and amniotic fluid prostaglandins (P <.05) but not interleukin-8 or interleukin-1 receptor antagonist. Amniotic fluid interleukin-6 and maternal and fetal cortisol, dehydroepiandrosterone sulfate, and estradiol concentrations were reduced by dexamethasone (P <.05), whereas tumor necrosis factor-alpha levels and leukocyte counts were attenuated by interleukin-10 treatment (P <.05). An inverse relationship was noted between amniotic fluid interleukin-10 concentrations and interleukin-1beta-induced uterine activity (r = -0.74, P <.05). CONCLUSION: Dexamethasone and interleukin-10 exert similar inhibitory effects on interleukin-1beta-induced uterine activity, which appears to be mediated by a decrease in prostaglandin production. Reduced estrogen biosynthesis or suppression of tumor necrosis factor-alpha and leukocyte migration may contribute to the tocolytic actions of dexamethasone and interleukin-10, respectively. Dexamethasone and interleukin-10 are likely to be useful adjuncts in the treatment of preterm labor that is associated with inflammation or infection.     

Mechanisms of matrix metalloproteinase-9 and matrix metalloproteinase-2 inhibition by N-acetylcysteine in the human term decidua and fetal membranes

OBJECTIVE: The purpose of this study was to evaluate the effect of N-acetylcysteine on the activity and secretion of the matrix metalloproteinases in the decidua, amnion, and chorion and the secretion of the tissue inhibitor of matrix metalloproteinase-1. STUDY DESIGN: Samples from eight nonlaboring women were taken at elective cesarean section and incubated in an in vitro organ culture in the absence or presence of N-acetylcysteine. Matrix metalloproteinase-2 and matrix metalloproteinase-9 activity was measured with the use of gel zymography. Western blot analysis was used to measure matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase-1 secretion. Data were analyzed with the paired Student t test. RESULTS: N-acetylcysteine had a direct inhibitory effect on matrix metalloproteinase-2 and matrix metalloproteinase-9 activity, regardless of tissue origin, starting at 1.0 mmol/L. In cultured media, 20 mmol/L N-acetylcysteine inhibited matrix metalloproteinase-2 and matrix metalloproteinase-9 activity in all three tissues. A differential response was demonstrated for matrix metalloproteinase-2 secretion, depending on the tissue that was studied. Its secretion was decreased in decidua at 10 mmol/L and 20 mmol/L; in amnion, the secretion was inhibited at 0.1 mmol/L and not affected at all in chorion. Matrix metalloproteinase-9 secretion was not affected in a statistically significant manner in any tissue. In the chorion, matrix metalloproteinase-9 showed a trend toward increased secretion. Tissue inhibitor of matrix metalloproteinase-1 secretion significantly decreased in the decidua at 20 mmol/L. CONCLUSION: N-acetylcysteine, at higher concentrations, has an inhibitory effect on matrix metalloproteinase-2 and matrix metalloproteinase-9 activity, regardless of the tissue origin and the differential effect on secretion depending on the tissue and N-acetylcysteine concentration.     

Stimulation of ovarian tumor cell proliferation with monocyte products including interleukin-1, interleukin-6, and tumor necrosis factor-alpha.

OBJECTIVE: We investigated whether monocyte-derived factors could stimulate the growth of ovarian cancer cells. STUDY DESIGN: Human peripheral blood monocytes or human monocyte-like cell lines THP-1 and U-937 were cultured with or without macrophage colony-stimulating factor, lipopolysaccharide, or phorbol myristate acetate. Culture supernatants or recombinant cytokines were assayed for growth stimulation of ovarian cancer cell lines by tritium-thymidine incorporation and direct cell counts followed by statistical analysis with Student t test. RESULTS: Conditioned medium from peripheral blood monocytes or from THP-1 or U-937 cells stimulated ovarian cancer cell growth. Interleukin-1 alpha, tumor necrosis factor-alpha, and interleukin-6 also stimulated ovarian cancer cell growth, whereas macrophage, granulocyte, and granulocyte-macrophage colony-stimulating factor did not. Concentrations of tumor necrosis factor, interleukin-1, and interleukin-6 in conditioned medium could not account for all the growth stimulation, and activity remained after neutralization of tumor necrosis factor, interleukin-1, and interleukin-6 with antibodies. CONCLUSIONS: Interleukin-1, interleukin-6, tumor necrosis factor, and additional monocyte factor(s) could provide paracrine growth stimulation when monocytes are attracted to ovarian cancers that produce macrophage colony-stimulating factor.     

Functional role of matrix metalloproteinases in ovarian tumor cell plasticity

OBJECTIVE: We previously demonstrated that aggressive ovarian cancer cells are able to display in vitro vasculogenic mimicry, which is reflected by their ability to form vasculogenic-like networks in 3-dimensional cultures and to express vascular cell-associated markers. The goal of this study was to examine the functional role of specific matrix metalloproteinases in the formation of vasculogenic-like networks and extracellular matrix remodeling in vitro. We also investigated the clinical relevance of matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase in human ovarian cancers with evidence of tumor cell-lined vasculature. STUDY DESIGN: Ovarian cancer cells (A2780-PAR, SKOV3, and EG) were seeded onto separate 3-dimensional cultures that contained either Matrigel or type I collagen, in the absence of endothelial cells or fibroblasts. These cultures were treated with either chemically modified tetracycline-3 (general matrix metalloproteinase inhibitor), recombinant tissue inhibitor of metalloproteinase-1 or -2, or function-blocking antibodies to matrix metalloproteinase-2 or -9 or membrane type 1-matrix metalloproteinase. In addition, 78 invasive epithelial ovarian cancers were evaluated for expression of matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase and correlated with various clinical parameters. RESULTS: The aggressive ovarian cancer cells (SKOV3 and EG) were able to form in vitro vasculogenic-like networks and contract 3-dimensional collagen I gels, whereas the poorly aggressive A2780-PAR cell line did not. Chemically modified tetracycline-3 completely blocked the network formation. Blocking antibodies to matrix metalloproteinase-2 and membrane type 1-matrix metalloproteinase inhibited the formation of the vasculogenic-like networks and collagen gel contraction, but the antibody to matrix metalloproteinase-9 had no effect on network formation and minimal effect on gel contraction. Treatment of 3-dimensional cultures with tissue inhibitor of metalloproteinase-2 retarded the network formation and only small, partially developed structures were noted that did not form network connections. Tissue inhibitor of metalloproteinase-1 had no appreciable effect on the extent or efficiency of network formation. Human invasive ovarian cancers with evidence of tumor cell-lined vasculature were significantly more likely to have strong epithelial and stromal matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase expression (all probability values were <.05). CONCLUSION: Matrix metalloproteinase-2 and membrane type 1-matrix metalloproteinase appear to play a key role in the development of vasculogenic-like networks and matrix remodeling by aggressive ovarian cancer cells. Human ovarian cancers with matrix metalloproteinase overexpression are more likely to have tumor cell-lined vasculature. These results may offer new insights for consideration in ovarian cancer treatment strategies.     

Expression and regulation of interleukin-8 in human fallopian tubal cells

OBJECTIVE: The human fallopian tube creates the microenvironment for fertilization and early embryogenesis. Salpingitis may result in infertility and ectopic pregnancy by causing tubal blockage and hydrosalpinx. To better understand the relationship between infectious inflammation and tubal damage, we investigated the expression and regulation of interleukin-8 in human tubal epithelial and stromal cells in culture. STUDY DESIGN: Human fallopian tube epithelial and stromal cell cultures were used to measure interleukin-8 messenger RNA and interleukin-8 protein levels at basal conditions and after stimulation with interleukin-1alpha and tumor necrosis factor-alpha. Northern blot analysis and enzyme-linked immunosorbent assay were used to evaluate messenger RNA and protein levels, respectively. RESULTS: Tubal epithelial cells expressed high levels of interleukin-8 messenger RNA and secreted significantly more immunoreactive interleukin-8 into culture medium than did tubal stromal cells (2065 +/- 153 pg/mg vs 530 +/- 56 pg/mg of total protein, P <.01). Interleukin-1alpha and TNF-alpha treatments induced a concentration-dependent increase in interleukin-8 messenger RNA expression in both epithelial and stromal cells. However, at the protein level, although interleukin-1alpha and tumor necrosis factor-alpha treatments increased the secretion of interleukin-8 from stromal cells significantly, similar treatments had no effect on interleukin-8 secretion from epithelial cells. CONCLUSION: The expression of interleukin-8 in human tubal epithelial and stromal cells is different. Interleukin-8 expression of tubal epithelial and stromal cells in response to inflammatory cytokines such as interleukin-1alpha and tumor necrosis factor-alpha also varies. This may be important in the pathogenesis of salpingitis.     

Tumor necrosis factor-alpha and interleukin-1 beta production by human fetal Kupffer cells.

This study describes the isolation and characterization of human fetal Kupffer cells. We demonstrated that these cells have the potential to respond to cytokines and lipopolysaccharide with an increased production of tumor necrosis factor-alpha and interleukin-1 beta. Kupffer cells were characterized by: (1) morphologic characteristics after adherence to plastic, (2) staining for alpha-naphthyl acetate esterase, (3) immunofluorescence with monoclonal antibodies, and (4) phagocytosis of latex beads. More than 90% of the adherent cells were identified as macrophages. Kupffer cells cultured with lipopolysaccharide were able to produce interleukin-1 beta and tumor necrosis factor-alpha in a time- and dose-dependent fashion and maximal secretion was observed with the use of 10 micrograms of lipopolysaccharide per milliliter within 8 hours of treatment. We have demonstrated mature functional activity of human fetal Kupffer cells at an early gestational age (13 to 19 weeks) and discussed the roles that these cells may play in development and protection of the fetus.     

Increased tumor necrosis factor-alpha production after lipopolysaccharide stimulation of whole blood in patients with previous preterm delivery complicated by intra-amniotic infection or inflammation.

OBJECTIVE: To compare cytokine production after lipopolysaccharide stimulation of whole blood from women who were delivered of infants at term compared with women who were delivered of preterm infants with intra-amniotic evidence of infection or inflammation. STUDY DESIGN: Whole blood samples from 12 women who were not pregnant and who had previously had preterm deliveries before 32 weeks complicated by intra-amniotic infection or inflammation and samples from 12 age- and race-matched control subjects were stimulated with Escherichia coli lipopolysaccharide. Tumor necrosis factor-alpha and interleukin-6 levels were quantified at 6 hours and interleukin-10 at 24 hours by enzyme immunoassay. Results were compared with use of the Wilcoxon rank sum test. RESULTS: Tumor necrosis factor-alpha production was significantly higher in whole blood from women with histories of a preterm birth and intra-amniotic infection or inflammation (11,243 +/- 1030 pg/mL [mean +/- SEM]) compared with control subjects (3649 +/- 349 pg/mL) at a lipopolysaccharide concentration of 1 microg/mL (P =.002). There were no significant differences in interleukin-6 or interleukin-10 production. CONCLUSION: Women with previous early preterm deliveries who had evidence of intra-amniotic infection or inflammation had significantly higher tumor necrosis factor-alpha production after lipopolysaccharide stimulation of whole blood compared with women with previous term deliveries.     

Influence of cytokines on matrix metalloproteinases produced by fibroblasts cultured in monolayer and collagen gels.

BACKGROUND: Extracellular matrix metalloproteinases (MMPs) are crucial factors involved in connective tissue remodeling that accompanies ultraviolet radiation-induced actinic damage. This study investigated whether the cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and IL-10 modulate the expression of MMPs in cultured human newborn skin fibroblasts. METHODS: Different concentrations of TNF-alpha, IL-1 beta, and IL-10 were added to human dermal fibroblasts grown in monolayers or embedded in three-dimensional (3D) collagen gels, a model closer to skin. Supernatant from the fibroblast cell culture was collected 24 hours later. The concentrations of MMP-1 and MMP-3 were assaysed by enzyme-linked immunosorbent assay (ELISA) while the concentrations of MMP-2 and MMP-9 were analysed by zymography. RESULTS: Basal production of MMPs was significantly greater in fibroblasts grown in 3D gels than in cells grown as monolayers. TNF-alpha and IL-1 beta induced increases in the concentrations of MMP-1, MMP-3, and MMP-9, but not in MMP-2 or tissue inhibitor of matrix metalloproteinase (TIMP)-1 or -2. The inducibility of MMP secretion is more significant in 3D gels. IL-10 did not significantly modulate MMPs. CONCLUSION: This study demonstrated that basal concentrations of MMPs are higher in fibroblasts cultured in 3D gels and their response to cytokines is different to that of cells grown as monolayers. Cytokines can increase the collagenolytic and gelatinolytic activity involved in extracellular matrix remodeling and hence contribute to photoaging.     

Modulation of trophoblast function by tumor necrosis factor-alpha: a role in pregnancy establishment and maintenance?

OBJECTIVE: Trophoblast differentiation is a critical process for successful implantation and establishment of the human placenta. The aim of this study was to characterize the effect of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) on the expression of markers of trophoblast function and differentiation.STUDY DESIGN: Human cytotrophoblasts were stimulated with 1 and 10 ng/mL recombinant TNF-alpha or IL-6. Cell viability was determined and conditioned culture media was analyzed by gelatin zymography to assess protease secretion and by enzyme-linked immunosorbent assays to measure production of beta-human chorionic gonadotropin and oncofetal fibronectin. RESULTS: TNF-alpha increased secretion of urokinase-type plasminogen activator up to 3-fold of basal unstimulated production. Stimulation of cytotrophoblasts with this cytokine also inhibited beta-human chorionic gonadotropin secretion up to 75%. TNF-alpha did not modify the secretion of matrix metalloproteinase-9 and oncofetal fibronectin. IL-6 had no effect on these trophoblast differentiation markers. CONCLUSION: These results show that TNF-alpha stimulated cytotrophoblasts modulate the expression of differentiation markers, down-regulating the autocrine signals that promote syncytialization, and increasing their invasive capacity through up-regulation of proteases. We suggest that this regulatory mechanism of trophoblast function could play an important role during trophoblast implantation, in pregnancy failure and in the normal and pathologic rupture of fetal membranes.     

MMP-9 mRNA as a therapeutic marker in acute and chronic stages of arthritis induced by type II collagen antibody.

BACKGROUND/PURPOSE: Antibodies against type II collagen (anti-CII) are arthritogenic and central to the initiation of the disease. An animal model of collagen type II-specific monoclonal antibody-induced arthritis (CAIA) has been used for the evaluation of various therapeutic effects in rheumatoid arthritis (RA). We aimed to measure the expression of matrix metalloproteinase (MMP)-9 (gelatinase B), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in the acute and chronic stages of the CAIA model for application as a therapeutic marker. METHODS: A commercially available antibody cocktail containing four monoclonal anti-type II collagen antibodies were injected into 6 to 8-week-old male BALB/c mice (n=20) and 50 microL lipopolysaccharide was injected 3 days later. The clinical manifestations of RA were recorded and scored at 10 days (acute stage) and 21 days (chronic stage). Then the mice were sacrificed for histologic analysis of the inflamed footpad and gene expression of IL-1beta, TNF-alpha and MMP-9 by ELISA and quantitative polymerase chain reaction amplification. RESULTS: Marked inflammation was found in the limb joints of mice at 10 days. Both IL-1beta and MMP-9 expression played a central role in the inflammatory reaction in the acute stage. The expression level of MMP-9 mRNA remained high in the chronic stage of CAIA, but that of IL-1beta mRNA was unexpectedly negligible; the serum level of TNF-alpha in CAIA was undetectable in the acute stage. The expression level of TNF-alpha mRNA was also lower than IL-1beta and MMP-9 in the acute inflammatory stage. CONCLUSION: The CAIA model is a fast and highly replicable model of RA. MMP-9 and IL-1beta were highly expressed in the acute stage of CAIA. It is suggested the MMP-9 mRNA level is a suitable marker for both acute and chronic stage, whereas IL-1beta is a marker only for the acute stage of the CAIA murine model.