Porcine spermatozoa inhibit post-breeding cytokine induction in uterine epithelial cells in vivo

Early endometrial cytokine responses after exposure to various inseminate components were investigated for a better understanding of the immunological reactions occurring in the porcine uterus after insemination. Baseline values were established for the mRNA concentrations of GM-CSF, IL-6, IL-10, CXCL8 (interleukin-8), Tumour Necrosis Factor α (TNF-α), TGF-β, cyclooxygenase-2 (COX-2) and arachidonate 5-lipooxygenase (ALOX-5) in periovulatory uterine endometrial tissue using quantitative RT-PCR. Synchronized gilts were inseminated with spermatozoa diluted either in the semen extender Androhep™ or seminal plasma. Uterine infusions of media without spermatozoa were used as controls. Three hours after insemination sows were slaughtered and the expression of the above mentioned cytokines was measured in uterine epithelial cells. Simultaneously, the influx of polymorphonuclear neutrophilic (PMN) granulocytes into the uterus was quantified. Compared to baseline values seminal plasma (SP) and Androhep™ (AH) respectively, if used alone, caused a significant increase in mRNA concentrations of IL-10 (SP: 1.5-fold), TGF-β (AH: 1.5-fold), CXCL8 (AH: 7.1-fold), TNF-α (AH: 1.9-fold) and COX-2 (AH: 7-fold). Surprisingly, in the presence of spermatozoa, none of the tested cytokines revealed mRNA concentrations higher than baseline values. The number of immigrated, intra-luminal PMN correlated only with mRNA concentrations of CXCL8 in presence of Androhep™ (r = 0.51). None of the other cytokines tested seemed to be involved in the regulation of neutrophil recruitment. However, the most interesting result was the sperm-induced down-regulation in the expression of TNF-α, TGF-β, IL-10, CXCL8 and COX-2 to mRNA concentration levels similar to or even below baseline values. In conclusion the results show that CXCL8 contributes significantly to uterine PMN recruitment and indicate a so far underestimated role of porcine spermatozoa in the general regulation of the uterine post-mating inflammatory response.     

Effects of Moritella viscosa antigens on pro-inflammatory gene expression in an Atlantic salmon (Salmo salar Linnaeus) cell line (SHK-1)

Moritella viscosa is the causative agent of winter ulcer disease in salmonids reared in North-Atlantic countries. In this study the effects of selected M. viscosa antigens on cytotoxicity and pro-inflammatory gene expression in an Atlantic salmon (Salmo salar Linnaeus) macrophage-like cell line (SHK-1) were examined. SHK-1 cells were stimulated with live and heat-killed bacterial cells, extracellular products (ECP) and an extracellular vibriolysin, termed MvP1. Following incubation, cytotoxicity and expression levels of interleukin-1β (IL-1β) and interleukin-8 (IL-8) were examined at different time points. Both live M. viscosa cells and ECP were cytotoxic, but neither heat-killed cells, nor the MvP1 peptidase caused cell death. Expression levels of both IL-1β and IL-8 increased significantly after stimulation with live cells, but heat-killed cells only caused increased IL-8 expression. ECP did not affect IL-1β expression, but did stimulate IL-8 expression. The isolated MvP1 peptidase stimulated both IL-1β and IL-8 expression at the highest concentration tested. This study reveals a difference in the induction of pro-inflammatory gene expression in salmon SHK-1 cells between live and heat-killed M. viscosa cells, and also that an unknown secreted factor is the main stimulant of IL-β and IL-8 expression.     

Leukotriene D4-induced adhesion of Caco-2 cells is mediated by prostaglandin E2 and upregulation of alpha2beta1-integrin

Cell-cell and extracellular matrix adhesions play important roles in the progression of cancer. We investigated the involvement of the inflammatory mediator leukotriene D4 (LTD4) in the regulation of cell-matrix adhesion of colon cancer (Caco-2) cells. We observed that LTD4 acted via its CysLT1 receptor in these cells to induce increased adhesion to collagen I. LTD4 also enhanced the activation and expression of alpha2beta1-integrins on the cell surface, which we found to be responsible for mediating the increased adhesion to collagen I. LTD4 simultaneously augmented expression of the prostaglandin-generating enzyme cyclooxygenase-2 (COX-2) and increased prostaglandin E2 (PGE2) production in Caco-2 cells. The adhesive capacity of the Caco-2 cells was reduced by specific inhibition of COX-2 and was subsequently restored by PGE2, but not by LTD4. A selective PGE2 receptor antagonist abolished the increased adhesion and the augmented alpha2beta1-integrin expression induced by both PGE2 and LTD4. Summarizing, the inflammatory mediator LTD4 regulates the adhesive properties and migration of the Caco-2 cell line by upregulating COX-2 and stimulating PGE2-induced expression of alpha2beta1-integrins. This suggests that inflammatory mediators such as LTD4 can be involved in the dissemination and survival of colon cancer cells.     

Role of prostaglandin H2 synthase 2 in murine parturition: study on ovariectomy-induced parturition in prostaglandin F receptor-deficient mice

To determine the prostaglandin (PG) H2 synthase (generally referred to as cyclooxygenase [COX]) isozyme responsible for producing uterotonic PGs during parturition, we used PGF2alpha receptor-deficient mice, which exhibit parturition failure due to impaired withdrawal of serum progesterone at term. On ovariectomy-induced parturition in these mice, uterine COX-2 mRNA expression was drastically induced in the myometrium, whereas COX-1 mRNA expression in the endometrial epithelium decreased. The concomitant administration of progesterone with ovariectomy resulted in a delay in parturition and the disappearance of both the increase in COX-2 mRNA and the decrease in COX-1 mRNA. Thus, the expression of myometrial COX-2 and the occurrence of parturition are closely associated in this model. Furthermore, administration of the COX-nonselective inhibitor, indomethacin, or the COX-2-selective inhibitor, Dup-697 or JTE-522, effectively delayed ovariectomy-induced parturition in these mice. These findings suggest that COX-2-derived PGs contribute to the onset of parturition after the decrease in serum progesterone level.     

Interleukin-1β-stimulated PGE2 production from early first trimester human decidual cells is inhibited by dexamethasone and progesterone

Cytokines are known to increase the production of prostaglandins by human decidual cells, but negative regulators have not been identified. We have examined the effects of dexamethasone and progesterone on prostaglandin (PG) E2 synthesis by cultured human first trimester decidual cells. The numbers of cyclooxygenase (COX) enzyme positive cells were visualised by immunocytochemistry, using antibodies specific for COX-1 and COX-2. Interleukin-1β stimulated the production of prostaglandins E2 and F2α dose-dependently, and this was associated with increased numbers of COX-2 positive cells. Progesterone (10⁻⁷−10⁻⁶ M) and dexamethasone (10⁻⁷−10⁻⁶ M) inhibited basal and interleukin-1β-stimulated prostaglandin production, and decreased the numbers of COX-2 positive cells. Neither interleukin-1β nor the steroids affected numbers of COX-1 positive cells. COX-2 seems to be the main enzyme controlling the synthesis of PGE2 by human decidual cells, and may be negatively regulated by progesterone.     

Combination of Tumor Necrosis Factor Alpha and Interferon Alpha Induces Apoptotic Cell Death through a c-myc–Dependent Pathway in p53 Mutant H226br Non–Small-Cell Lung Cancer Cell Line

We investigated the role of wild-type p53 and c-myc activity in apoptosis induced by a combination of natural human tumor necrosis factor alpha (TNF-α) and natural human interferon alpha (IFN-α). Studies were performed with two human non–small-cell lung cancer cell lines, H226b, which has wild-type p53, and H226br, which has a mutant p53. The combination of IFN-α and TNF-α significantly inhibited cell growth and induced apoptotic cell death of both H226b and H226br, compared with IFN-α or TNF-α alone. Treatment with one or both cytokines did not affect the expression level of p53 in both cell lines. These results suggest that the combination of IFN-α/TNF-α induces apoptotic cell death through a p53- independent pathway. The c-myc oncogene is known to be involved in apoptosis induced by TNF. Antisense c-myc oligonucleotides have been reported to modulate cell growth or apoptosis in several cell lines. Antisense oligodeoxynucleotides were added to the culture of H226br cells before the addition of IFN-α/TNF-α. Antisense c-myc inhibited IFN-α/TNF-α cytotoxicity and apoptotic cell death. In conclusion, this study provides support for the speculation that TNF-α/IFN-α induce apoptosis through a c-myc–dependent pathway rather than a p53-dependent pathway.     

Interaction between uterine PGE and PGF2α production and the nitridergic system during embryonic implantation in the rat

Embryonic implantation is a complex process in which both maternal andembryonic signals are involved. In the present study, we evaluated changes in uterine prostaglandins production and nitric oxide synthase (NOS) activity during the course of early pregnancy and their interaction during implantation in rats. Uterine phospholipase A₂ (PLA₂) activity is increased on days 5 (day of ovoimplantation) and 6, compared to preimplantation days (3 and 4). This enhanced activity might be responsible for the observed increase in uterine PGE and PGF_2α production observed on day 5 of pregnancy, which induces endometrial vascular permeability and decidualization. When embryo access to the uterus is impaired, the increase of PG production is suppressed. During postimplantation, PGE levels return to preimplantation values, while PGF_2α decreased with respect to preimplantation values. Uterine NOS activity is also increased on day 4 and reaches a maximum on day 5, with a profile similar to PGE and PGF_2α Dexamethasone administered in vivo decreased uterine NOS activity on day 4 of pregnancy but not on day 5, suggesting the presence of at least two types of NOS enzymes in the early days of pregnancy. A competitive inhibitor of NOS, L-NAME (600 and 1000 μM) induced a decrease in PGE and PGF_2α production in uterine tissue on day 5 of pregnancy. These results suggest the existence of a physiologically relevant nitridergic system which modulates prostaglandin production in the rat uterus during embryonic implantation.     

Transforming growth factor-β enhances secretory component and major histocompatibility complex class I antigen expression on rat IEC-6 intestinal epithelial cells

Transforming growth factor-β (TGF-β) has been implicated as having a role in inflammatory responses by inducing cellular infiltration and the release of inflammatory cytokines. In this study, the IEC-6 rat intestinal epithelial cell line was used as a model to assess the effect of TGF-β₁ on the expression of various plasma membrane determinants. TGF-β₁ induced a dose-dependent increase in the percentage of cells expressing surface secretory component (SC) and class I major histocompatibility (MHC) antigens. However, the expression of class II MHC was unaffected. In contrast, epidermal growth factor had no effect on any of the surface proteins studied. The TGF-β₁-enhanced expression of SC was accompanied by an enhanced binding of polymeric, but not monomeric, immunoglobulin A (IgA). Preincubation of the TGF-β₁-treated cells with an anti-human β-galactosyltransferase (β-GT) antiserum did not block the binding of the anti-SC antibody, indicating that the TGF-β-induced increase in SC staining was due to SC expression and not the polymeric immunoglobulin-binding enzyme, β-GT. These results indicate that TGF-β₁ may be important in immune functions involving intestinal epithelial cells by enhancing the expression of surface class I MHC antigens and SC, a protein responsible for the transport of polymeric IgA into the intestinal lumen.     

IGF-II Enhances Trichostatin A-Induced TGFβ1 and p21 Expression in Hep3B Cells

Cell growth and division are controlled through the actions of cyclin-dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CKIs). Treatment of cell lines with Trichostatin A leads to induction of one of these CKIs, p21, and growth arrest. Induction of p21 can also occur through the actions of TGFβ1. Latent TGFβ1 can be activated by the M6P/IGF2R. In the present study we have examined the effect of TSA on members of the IGF axis, the CKIs p21 and p27, and also TGFβ1 in Hep3B cells. The only member of the IGF axis to be affected by treatments was IGF2. Expression of another gene from the same chromosomal location, H19, was also affected. TGFβ1 expression was greatly enhanced by TSA. In addition, both CKIs, p21 and p27, were upregulated by TSA. Effects of adding IGF-II or TGFβ1 to TSA-treated cells on p21 induction were examined. The results show that the induction of p21 by TSA can be modulated by additions of IGF-II whereas addition of TGFβ1 affects its own expression but not p21. In conclusion, the results indicate that the induction of p21 and cell growth arrest caused by Trichostatin A may involve multiple signaling pathways.     

Characterization of in vitro proteolytic processing of β-endorphin by reversed-phase HPLC

In vitro, central and peripheral proteolytic processing of β-endorphin by membrane-bound enzymes results in the formation of specific active fragments that have been recently shown to function in behavior, intestinal motility and in the central control of urinary bladder activity. A high resolution, reversed phase high performance liquid chromatography system capable of separating 28 β-endorphin related fragments simultaneously was used to study the time-course processing of β-endorphin by membrane associated peptidases in the brain and regions of the small intestine. The hypothesis we tested was that a homeostatic balance between α- and γ-type endorphins exists in these tissues. The results of the study show that the rate and quantity of fragments produced between the mucosa and nerve-muscle regions of the small intestine are significantly different. Metabolic rates, pattern, and the ratio of α/γ-type endorphins in the brain were very similar to the nerve-muscle region of the small intestine. This suggests that β-endorphin processing to active fragments is occurring at the nerves of the small intestine and that a specific and similar balance of α/γ-type endorphin exists in the brain and gastrointestinal system at neutral pH.     

Effects of protein kinase C activation on prostaglandin production and cyclooxygenase mRNA levels in ovine astroglia

We examined effects of protein kinase C (PKC) activation by phorbol dibutyrate (PDB) on prostaglandin production in astroglia. Astroglia were cultured from sheep fetal cortex and grown in Eagle's basal media supplemented with 10% fetal calf serum (BME-C). Prostaglandin F_2a (PGF_2α) levels in media were determined at 2–24 hours after exposure to PDB. PDB increased production of PGF_2α at 10⁻⁸M and 10⁻⁶M. In addition, PDB increased the ratio of membrane to cytosolic PKC. Coapplication of H7 [1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine] (10⁻⁴M) with PDB (10⁻⁶M) inhibited PDB-induced PGF_2a production. To investigate the role of protein synthesis in increased prostaglandin production by PDB, astroglia were coincubated with actinomycin D (1 mg/ml) or cycloheximide (10 mg/ml). At 4 hrs, both actinomycin D and cycloheximide inhibited increases in PGF_2a in response to PDB application. In addition, COX-2 mRNA levels and COX activity levels were examined. PDB increased COX-2 mRNA levels by 2 hours, and COX activity tripled after 12 hr exposure to PDB. In addition, the increase in COX activity was blocked by cycloheximide. In summary, PKC activation promotes enhanced prostaglandin production via an increase in COX synthesis.     

Regulation of COX-2 expression by miR-146a in lung cancer cells

Prostaglandins are a class of molecules that mediate cellular inflammatory responses and control cell growth. The oxidative conversion of arachidonic acid to prostaglandin H₂ is carried out by two isozymes of cyclooxygenase, COX-1 and COX-2. COX-1 is constitutively expressed, while COX-2 can be transiently induced by external stimuli, such as pro-inflammatory cytokines. Interestingly, COX-2 is overexpressed in numerous cancers, including lung cancer. MicroRNAs (miRNAs) are small RNA molecules that function to regulate gene expression. Previous studies have implicated an important role for miRNAs in human cancer. We demonstrate here that miR-146a expression levels are significantly lower in lung cancer cells as compared with normal lung cells. Conversely, lung cancer cells have higher levels of COX-2 protein and mRNA expression. Introduction of miR-146a can specifically ablate COX-2 protein and the biological activity of COX-2 as measured by prostaglandin production. The regulation of COX-2 by miR-146a is mediated through a single miRNA-binding site present in the 3′ UTR. Therefore, we propose that decreased miR-146a expression contributes to the up-regulation and overexpression of COX-2 in lung cancer cells. Since potential miRNA-mediated regulation is a functional consequence of alternative polyadenylation site choice, understanding the molecular mechanisms that regulate COX-2 mRNA alternative polyadenylation and miRNA targeting will give us key insights into how COX-2 expression is involved in the development of a metastatic condition.