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Objective: Endometriosis, a common disease among women of reproductive age, is characterized by the presence of endometrial-like tissue outside the uterus. TNF-alpha induces IL-8 production in endometriotic cells through nuclear factor-kappaB (NF-kappaB) activation. Thalidomide (Thal) inhibits inflammation by down-regulating the expression of proinflammatory cytokines in tumor cells and inflammatory cells. However, the mechanism of Thal action in human endometriotic stromal cells has not yet been elucidated., Main Outcome Measures: We examined whether Thal abrogates TNF-alpha-induced up-regulation of IL-8 expression in endometriotic stromal cells., Results: Here, we show 1) that treatment of endometriotic stromal cells with TNF-alpha increased the expression of phosphorylated IkappaBalpha and degradation of total IkappaBalpha, which in turn activates NF-kappaB; 2) Thal significantly inhibits the TNF-alpha-induced expression of phosphorylated IkappaBalpha and degradation of IkappaBalpha; 3) TNF-alpha activation induced increased nuclear translocation of NF-kappaB, which was inhibited by pretreatment with either Thal or N-tosyl-L-phenylalanine chloromethyl ketone, an NF-kappaB inhibitor. Thal did not enhance the N-tosyl-L-phenylalanine chloromethyl ketone's action; and 4) Pretreatment with Thal reduced TNF-alpha-induced IL-8 protein production as well as mRNA expression., Conclusion: The current study showed for the first time that Thal treatment attenuated the expression of IL-8 by reducing TNF-alpha-induced NF-kappaB activation.

The aim of the present study was to elucidate functions of the interleukin (IL)-8 at ovulation and luteinization in vivo. To compare the morphological differences between human chorionic gonadotropin (hCG) and IL-8 stimulation, scanning electron microscopy was employed to study rat ovarian vascular corrosion casts. Follicular growth and increased capillary vessel densities around the follicles were seen in vascular corrosion casts after IL-8 injection, similar to the result of hCG administration. This result indicated that exogenous IL-8 could play a role in the neovascularization during follicular development as an angiogenetic factor. Many fenestrations were observed in the vascular endothelium by hCG administration. In contrast, no fenestrations were observed with IL-8 injection, indicating that IL-8 may not be sufficient to increase the vascular permeability directly. Although germinal vesicle breakdown (GVBD) occurred at rates of 82% after the hCG injection, only 20% GVBD was observed after the IL-8 injection. The present study indicated that IL-8 might have important effects on rat follicles at ovulation and luteinization via vascularization in a similar manner to hCG. However, IL-8 was not effective on vascular permeability and oocyte maturation, which were different from hCG. Thus, we can conclude that IL-8 can participate in follicular development in part and may play important roles in ovulation and luteinization as one of some mediators induced by endogenous luteinizing hormone.

Interleukin 8 (IL-8) is a neutrophil chemoattractant/activating factor that plays a role in the ovarian physiology leads to investigate the effects of IL-8 on follicular maturation. Experiments were conducted using suppositories containing 100 ng, 200 ng, 400 ng IL-8, 500 microl Witepsol-base (control), human menopausal gonadotropin (im) and conjugate of fluorescein isithiocyanate-labelled IL-8. The levels of IL-8 in ovarian fluid were also measured. Histology of ovaries treated with 200 ng IL-8 showed large antral follicles filled with follicular fluid. The theca layer was divided into an interna and an externa with large extracellular spaces. The granulosa cells were loosened and appeared to be detaching from the granulosa layer. Neutrophils were localized predominantly in the theca and medulla (P<0.0001, P<0.004), and relative collagen concentration was significantly decreased in ovaries of 200 ng IL-8 (P<0.0001) compared with controls. The IL-8 was detected in ovarian fluid after 6 h (P<0.0001), 12 h (P<0.001), and 18 h (P<0.01) compared with 0 h. Fluorescein isithiocyanate-labelled IL-8 conjugate was seen in the follicular wall and endometrium. We conclude that pharmacological dosage of exogenous IL-8 exerts an effect on follicular maturation through granulocyte chemotaxis and activation.

Cervical ripening is a cytokine-triggered process with substantial remodelling of the cervical extracellular matrix. Interleukin-8 (IL-8) is an important cytokine in cervical maturation. Glycosaminoglycans are also included in this process, but their role in not clearly understood. The effects of heparan sulphate (HS), hyaluronic acid (HA), IL-8, HS + IL-8 and HA + IL-8 on biochemical properties of the cervix were examined in non-pregnant rabbits. The changes in vascular pattern with collagen structure of the cervices and immunohistochemical studies, together with the relative collagen concentrations, were determined. A reduction in relative collagen concentration was significant after HS + IL-8, IL-8 and HA + IL-8 treatment (all P < 0.0001). Gel electrophoresis analysis showed that IL-8 bound preferentially to HS than to HA. Neutrophils were significantly increased in number (P < 0.0001) and located predominantly beneath the glandular epithelium and around the blood vessels after HS + IL-8 treatment. HS + IL-8 treatment caused cervices to increase their water content and become oedematous. The collagen fibres were considerably dissociated, the interfibrillar spaces markedly dilated, and the blood vessels notably increased and dilated. We conclude that binding to HS enhances the activity of IL-8 in inducing cervical maturation.

The aim of this research was to study the effect of exogenous interleukin (IL)-8, IL-1 and IL-8 + IL-1 on uterine contractions in rabbits. Four equal groups of non-pregnant rabbits (n = 24) were investigated using either placebo or experimental drugs in the form of vaginal suppositories. The suppositories contained human recombinant IL-8 (200 ng), IL-1 (200 ng), IL-8 (200 ng) + IL-1 (200 ng) or vehicle Witepsol base, 500 microliters). Subsequently, the plasma concentration of prostaglandin (PG) E2 was estimated 3 h after the last dose of treatment. Neutrophil infiltration in the endometrial tissue was studied with anti-rabbit RT2 staining. Suppositories with IL-1 and IL-8 + IL-1 produced contractile responses with increased frequency (P < 0.003, P < 0.0005) and amplitude (P < 0.0001) in vivo, compared with vehicle. IL-1 and IL-8 + IL-1 also caused similar contractile effects with increased frequency (P < 0.01, P < 0.0007) and amplitude (P < 0.0001) in an in-vitro experiment than vehicle. The frequency and amplitude of uterine contractions were more significant with IL-8 + IL-1 than that of IL-1, both in vivo (P < 0.002, P < 0.05) and in vitro (P < 0.005, P < 0.01). IL-8 did not induce any contractions. Prostaglandin concentration was increased approximately 8-fold with IL-8 + IL-1 (P < 0.0001) and 2.5-fold with IL-1 treatment (P < 0.0001). Neutrophil numbers were significantly increased with IL-8 + IL-1 > IL-8 > IL-1 (P < 0.002, P < 0.0003 and P < 0.008) compared with vehicle. Our data suggest that IL-8 stimulates IL-1-induced uterine contractions through PGE2 production and could be an important process during labour and delivery.

We investigated the effects of dehydroepiandrosterone sulfate (DHA-S) on the production of interleukin-8 (IL-8) and expression of the interleukin-8 receptor (IL-8 R) in human cervical tissue. DHA-S increased the levels of IL-8 in cultured human cervical fibroblasts and in the supernatant in a time- and dose-dependent manner. DHA-S induced IL-8 and IL-8 R expression in human cervical fibroblasts and human pregnant cervical tissue at term in a dose-dependent manner. In addition, it induced the expression of IL-8 R in an explant culture of human cervical tissue and cultured human cervical fibroblasts in a time- and dose-dependent manner. However, dehydroepiandrosterone (DHA) only slightly induced the production of IL-8 and the expression of IL-8 R in the same cells and tissue. These results suggest that DHA-S up-regulates the autocrine system of IL-8 through the expression of IL-8 R.

Although the effects of inflammatory cytokines such as interleukin (IL)-1 and tumour necrosis factor (TNF)-alpha on ovulation have been investigated, there are few reports concerning the effect of IL-8 in the ovary. We examined the localization of IL-8 in the human ovary, and also investigated the number of IL-8 staining cells in a section of 1 mm2. The number of IL-8 staining cells was 10 +/- 4.8 in the proliferative phase, 2.0 +/- 1.9 in the secretary phase, and 2.4 +/- 2.2 in the postmenopausal phase respectively. The increased number of IL-8 staining cells could be shown in the human ovarian medulla/stroma during the proliferative phase. By serial section analysis of the ovary, the localization of IL-8 corresponded with mast cells. When immature rats were injected with pregnant mare serum gonadotrophin (PMSG) followed by an i.p. injection of human IL-8, ovarian vasodilatation and follicular growth were observed. Exogenous IL-8 induced a similar increase in follicular growth to that produced by the luteinizing hormone (LH) surge. At 6 h after IL-8 (20 microg) i.p., the follicular size was increased to almost the same size as human chorionic gonadotrophin (HCG) i.p. However, at 24 h after IL-8 i.p., the follicular size decreased. The size of follicular growth was almost the same size as control when <2 microg IL-8 was injected. At 6 h after IL-8 (10 microg) local injection, the follicular size in rats was similar to that observed with IL-8 (20 microg) i.p. We conclude that IL-8 is one of the important cytokines in the ovulatory process.

During pregnancy, hyaluronic acid (HA) concentration in the human cervix is very low, but increases rapidly at the onset of labour. HA has a high affinity for water molecules and hence can maintain tissue hydration. HA can stimulate collagenase production in rabbit cervix, and also stimulates migration and function of polymorphonuclear leukocytes in the tissues. It is an endogenous regulator of interleukin-1 (IL-1). We hypothesized that HA plays an essential role during cervical ripening. The effect of exogenous application of HA (20 mg) on non-pregnant and pregnant (day 23) rabbit cervices was compared with controls. HA induced cervical ripening in both pregnant and non-pregnant animals, and cervical water content was significantly increased. Tissue collagen was markedly decreased. The localization and distribution of HA and HA receptor CD44 was determined in non-pregnant and pregnant human cervical connective tissue using biotinylated HA binding protein and CD44 monoclonal antibodies. Both were widely distributed in the connective tissues, especially around the blood vessels and cervical glands. The effect of IL-8 (50, 100, 150 and 200 ng/ml) on HA production and hyaluronidase (HAase) activity was investigated in cultures of lower uterine segment collected during elective Caesarean sections. HA production was stimulated in a dose-dependent manner; there was no effect on hyaluronidase activity. HA administration (0.5, 1 and 2 mg/ml) stimulated the activities of collagenase and gelatinase together with IL-8 production in the culture supernatants. Thus HA may play an important role in cervical ripening, being involved in the regulation of cervical tissue water content, collagenolytic enzymes and cytokines.

Objective: Interleukin-8 (IL-8) is known to play a crucial role in human parturition. We aimed to study the effect of mechanical stretching on the expression of IL-8 in fetal membranes (amniochorion) and decidua., Study Design: We examined the expression of IL-8 and its receptor in fetal membranes (amniochorion) and decidua by immunohistochemical staining. Also, we studied the synthesis of IL-8 messenger RNA (mRNA) in the fetal membranes before and after stretching., Results: We found that mechanical stretching within physiological limit increased IL-8 messenger RNA (mRNA) synthesis in fetal membranes and decidua in a time- and load-dependent manner. Application of mechanical force led to markedly increased staining of IL-8 receptor in decidual cells but not in amnion or chorion cells., Conclusion: These results suggested that mechanical stretching was a candidate for one of the signals important for production of IL-8 in fetal membranes and decidua and probably for initiation of a cytokine network at amniochorio-decidual interface through increased expression of IL-8 receptors.

Background: Interleukin-8 (IL-8) has an essential role in the mechanism of parturition. IL-8 induces its effect through specific cell binding receptors. The main purpose of this research was to elucidate the presence of IL-8 receptors in the gestational tissues and their relation to parturition., Materials and Methods: Fetal membranes, placenta, umbilical cord and myometrial samples were collected before the initiation of labor during elective Cesarean sections (n = 15). Similar biopsies (except myometrial samples) were also gathered after normal vaginal delivery (n = 15). Immunohistochemical staining for IL-8 receptors type I and II was carried out for the fresh frozen sections of all samples. Immunoreactive score (IRS) was calculated for the stained sections., Results: IL-8 Receptors type I and II were recognized in the fetal membranes, placenta, umbilical cord and myometrial samples collected from patients before the initiation of labor. The staining of amnion and placentas were found to be more intensified in the samples collected after vaginal delivery compared to those from elective cesarean sections and before initiation of labor., Conclusions: Increased expression of IL-8 receptors after initiation of labor may indicate that IL-8 receptors might have a role during parturition.

The aim of this research was to study and compare the mechanism of action of interleukin (IL)-8, IL-1beta dehydroepiandrosterone sulphate (DHEA-S) and prostaglandin (PG)E2 on the cervix. Five equal groups of pregnant rabbits (n = 45) were tested by either placebo or tested drugs in the form of vaginal suppositories once daily for 3 days. The suppositories contained human recombinant IL-8 (100 ng), IL-1beta (200 ng), DHEA-S (10 mg) or PGE2 (1 mg). All rabbits were tested by one dose, two doses or three doses. Consistency, dilatation and water contents were estimated 24 h after the last dose of treatment. Leukocyte infiltration of the cervices was studied after staining the cervical tissue sections with antirabbit RT2 monoclonal antibodies. Relative collagen concentration was assessed after staining with Picrosirius Red. Collagenase, gelatinase and elastase activities were measured in 100 mg of homogenized cervical connective tissue. Water contents were significantly increased in all tested cervices. Neutrophil numbers were increased in IL-8 and IL-1beta groups after the second dose of treatment (P < 0.0005 and 0.001 respectively). In the PGE2 group, neutrophils were increased after the third dose of treatment, whereas in DHEA-S group no significant changes were observed. Collagen content was significantly decreased in IL-8, IL-1beta and PGE2 groups after the first dose of treatment (P < 0.004, and 0.005 and 0.03 respectively). In the DHEA-S group, the decrease in collagen content occurred after the third dose (P < 0.05). Collagenase activity was markedly increased in IL-8, IL-1, and DHEA-S groups after the second dose of treatment (P < 0.001, 0.003 and 0.007 respectively). No significant increase in collagenase activity was found in PGE2 group. Gelatinase activity was significantly increased in IL-8, IL-1beta, PGE2 and DHEA-S groups after the second dose of treatment (P < 0.008, 0.01, 0.003 and 0.05 respectively). Also, elastase activity was increased after the second dose of treatment in all groups (P < 0.001, 0.001, 0.001 and 0.006 respectively). Our data suggest that ripening of the cervix in rabbit can be initiated by different mechanisms. Cytokines play a vital role in cervical ripening, especially IL-8 and IL-1. IL-8 is one of the factors which could ripen the cervix in a manner similar to the physiological process at term.

Objective: The aim of this study was to determine whether stretching of fetal membranes can increase interleukin-8 concentration and collagenase activity., Study Design: Strips of whole fetal membranes, amnion, or muscles of the lower uterine segment were stretched for 2 or 4 hours. Interleukin-8 and collagenase activity were measured in homogenized control and stretched samples. Immunohistochemical staining for interleukin-8 was carried out., Results: The interleukin-8 concentration increased significantly after the whole fetal membranes were stretched for 2 and 4 hours (p <0.0007 and 0.001, respectively). Also, stretching of amnion and muscles of the lower uterine segment for 2 and 4 hours increased the concentration of interleukin-8 significantly (p <0.0007 after 2 and 4 hours, respectively). Collagenase activity was significantly increased after stretching of amnion, amniochorion, and muscles of the lower uterine segment for 4 hours (p <0.0007, 0.006, and 0.0007, respectively). After stretching, samples were darkly stained for interleukin-8 compared with control nonstretched samples., Conclusion: Stretching of amnion, amniochorion, and muscles of the lower uterine segment increased interleukin-8 production and collagenase activity.

Dehydroepiandrosterone sulfate (DHA-S) is one of the successful drugs used to induce cervical ripening. There is growing evidence that interleukin-8 (IL-8) plays an essential role during cervical ripening. IL-8 was found to be hormonally controlled in the cervix. The aim of this research was to study the effect of DHA-S on the function of IL-8 on the cervix. Sixteen pregnant white New Zealand rabbits where tested either by placebo (adeps solidus, n = 4), DHA-S (10 mg, n = 4), IL-8 (100 ng, n = 4) or combination of DHA-S and IL-8 (n = 4). Collagen contents in the cervices were assessed histologically after staining with picrosirius red. The number of neutrophils in 5 random fields of the cervical tissue was counted in all groups. Collagenase, elastase and gelatinase were measured in those groups. The collagen content of the cervices was significantly decreased after treatment with DHA-S, IL-8 or a combination of both drugs compared to the control group (p < 0.004, 0.001 and 0.001, respectively). A maximal decrease in collagen content was found in the DHA-S + IL-8-treated group (71.5%). Neutrophil infiltration was significantly increased in the IL-8 and DHA-S + IL-8-treated groups (p < 0.0001). Neutrophil numbers in the DHA-S + IL-8-treated group were significantly higher than in the IL-8 group (p < 0.006). Collagenase activity was increased in the DHA-S, IL-8 and DHA-S + IL-8 groups compared to the control group (p < 0.02, 0.008 and 0.001, respectively). Elastase activity was also significantly increased in the same groups (p < 0.002, 0.005 and 0.001, respectively). A similar increment was found in gelatinase activity (p < 0.004, 0.0001 and 0.0001, respectively). DHA-S has a synergistic effect with IL-8 during cervical ripening.

Bestatin is an immunomodulatory peptide that stimulates the humoral and cell-mediated immune system. It also has an inhibitory effect on multiple aminopeptidases. Recently we found that aminopeptidase N inactivates interleukin-8 in vitro. Bestatin successfully suppresses the effect of aminopeptidase N on interleukin-8. During cervical maturation many biochemical changes occur including decrease in collagen concentration and increase in collagenase and elastase activities. Interleukin-8, which has a potent neutrophil chemotactic effect, was found to induce cervical ripening in rabbits. The combination of interleukin-8 with bestatin also induced cervical ripening by providing approximately regular levels of neutrophil numbers, collagenase, and elastase activities. We therefore suggest that this regulatory mechanism also takes place in vivo through the inhibitory effect of bestatin on aminopeptidase N.

Urinary trypsin inhibitor (UTI) is one of the Kunitz-type protease inhibitors in human. Little is known about its anti-inflammatory functions other than protease inhibition. We studied the effect of UTI on gene expression of interleukin-8 (IL-8), an inflammatory cytokine. UTI inhibited IL-8 gene expression induced by lipopolysaccharide (LPS) in HL60 cells. The IL-8 concentrations in the cells and medium after LPS stimulation increased time-dependently in the absence of UTI, but did not increase in the presence of UTI. On the other hand, UTI did not inhibit either the synthesis or the release of IL-8 induced by the calcium ionophore A23187. UTI inhibited increase of cytosolic Ca2+ stimulated by LPS but not by A23187. Our results suggest that the inhibition by UTI is due to its effect on the cell membrane involved in regulating Ca2+ influx.

Our aim was to study the effects of infection mediated by bacterial endotoxins (lipopolysaccharides LPS) and bacterial substrate (N-formyl-methionyl-leucyl-phenylalanine FMLP) on pathological cervical ripening, and compare their effects with the normal physiological mediator interleukin-8 (IL-8). Twenty non-pregnant rabbits were treated vaginally either by placebo, LPS, FMLP or IL-8 for 3 successive days. Water concentration was measured. Neutrophils were counted in 5 random fields of each specimen. Collagen and glycosaminoglycan concentrations were assessed histologically by using picrosirius red and alcian blue staining and optical density was calculated. LPS FMLP as well as IL-8 can induce cervical ripening. Water concentration was found to be significantly increased in these groups (p < 0.0001). Also, significant numbers of neutrophils were found to accumulate in the connective tissue of these cervices (p < 0.0001). Collagen concentration was significantly decreased in the cervices treated by LPS, FMLP and IL-8 (p < 0.0001). Glycosaminoglycan concentration was significantly increased in cervices treated by LPS, FMLI as well as IL-8 (p < 0.0001, 0.006 and 0.001 respectively). LPS, FMLP and IL-8 stimulate cervical ripening in non-pregnant rabbits. This ripening was mediated through the accumulated neutrophils in the connective tissue. IL-8 has a physiological type effect to induce ripening of the cervix.

Aminopeptidase (APN) was found to degrade interleukin-8 (IL-8) and inactivate its chemotactic activity. The chemotactic activity of IL-8 was decreased by APN or neutrophil plasma membranes dose- and time-dependently. The chemotactic activity was not inactivated in the presence of bestatin or WM15 monoclonal antibody. The expression of IL-8 was measured by flow cytometry. On lipopolysaccharide (LPS) stimulation, IL-8 expression increased for 60 min and then decreased markedly. In contrast, on treatment with LPS and bestatin, the expression of IL-8 increased continuously for at least 120 min. These results suggest that the expression and release of IL-8 from phagocytic cells are regulated by the proteolytic effect of APN on IL-8.

Objective: The purpose of this study was to determine whether cervical ripening can be induced in rabbits by interleukin-8., Study Design: Nonpregnant and pregnant rabbits were treated for 5 days with vaginal suppositories containing 100 ng of interleukin-8. Collagen and glycosaminoglycan concentration in the cervices were assessed histologically by picrosirius red and alcian blue, and the mean optical density was calculated. The mean neutrophil count in five random fields was calculated from each biopsy specimen., Results: Interleukin-8 induced softening and dilatation of the rabbit cervices. Water content was significantly increased (p < 0.0001 and p < 0.001, respectively). Cervical collagen concentration was found to be significantly decreased (p < 0.0004 and p < 0.0001, respectively). Glycosaminoglycan concentration was significantly increased in nonpregnant and pregnant cervices (p < 0.0009 and p < 0.1, respectively). The mean number of neutrophils was significantly increased (p < 0.0005 and p < 0.006, respectively)., Conclusion: Interleukin-8 can induce cervical ripening in nonpregnant and pregnant rabbits.