Progesterone-induced blocking factor differentially regulates trophoblast and tumor invasion by altering matrix metalloproteinase activity

Invasiveness is a common feature of trophoblast and tumors; however, while tumor invasion is uncontrolled, trophoblast invasion is strictly regulated. Both trophoblast and tumor cells express high levels of the immunomodulatory progesterone-induced blocking factor (PIBF), therefore, we aimed to test the possibility that PIBF might be involved in invasion. To this aim, we used PIBF-silenced or PIBF-treated trophoblast (HTR8/Svneo, and primary trophoblast) and tumor (HT-1080, A549, HCT116, PC3) cell lines. Silencing of PIBF increased invasiveness as well as MMP-2,-9 secretion of HTR8/SVneo, and decreased those of HT-1080 cells. PIBF induced immediate STAT6 activation in both cell lines. Silencing of IL-4Rα abrogated all the above effects of PIBF, suggesting that invasion-related signaling by PIBF is initiated through the IL-4Rα/PIBF-receptor complex. In HTR-8/SVneo, PIBF induced fast, but transient Akt and ERK phosphorylation, whereas in tumor cells, PIBF triggered sustained Akt, ERK, and late STAT3 activation. The late signaling events might be due to indirect action of PIBF. PIBF induced the expression of EGF and HB-EGF in HT-1080 cells. The STAT3-activating effect of PIBF was reduced in HB-EGF-deficient HT-1080 cells, suggesting that PIBF-induced HB-EGF contributes to late STAT3 activation. PIBF binds to the promoters of IL-6, EGF, and HB-EGF; however, the protein profile of the protein/DNA complex is different in the two cell lines. We conclude that in tumor cells, PIBF induces proteins, which activate invasion signaling, while—based on our previous data—PIBF might control trophoblast invasion by suppressing proinvasive genes.     

Effect of human serum on alkaline phosphatase induction in cultured human tumor cells

The continuous cell lines T 24 and HT-29, derived from human bladder and colon carcinomas, produce term-placental and intestinal alkaline phosphatase, respectively. Growth in hyperosmolar medium or exposure to prednisolone or sodium butyrate induces increased enzyme levels, and combinations of inducers elicit synergistic activity increases. The effect of the inducing agents is strikingly diminished when cells are grown in the presence of high concentrations of human serum, and the synergistic increases are essentially abolished. Major human serum protein fractions do not affect alkaline phosphatase induction.     

Effect of human serum on alkaline phosphatase induction in cultured human tumor cells

Summary The continuous cell lines T 24 and HT-29, derived from human bladder and colon carcinomas, produce term-placental and intestinal alkaline phosphatase, respectively. Growth in hyperosmolar medium or exposure to prednisolone or sodium butyrate induces increased enzyme levels, and combinations of inducers elicit synergistic activity increases. The effect of the inducing agents is strikingly diminished when cells are grown in the presence, of high concentrations of human serum, and the synergistic increases are essentially abolished. Major human serum protein fractions do not affect alkaline phosphatase induction.     

Increased mitochondrial palmitoylcarnitine/carnitine countertransport by flavone causes oxidative stress and apoptosis in colon cancer cells

Cancer cell metabolism is characterized by limited oxidative phosphorylation in order to minimize oxidative stress. We have previously shown that the flavonoid flavone in HT-29 colon cancer cells increases the uptake of pyruvate or lactate into mitochondria, which is followed by an increase in O₂^−.. production that finally leads to apoptosis. Similarly, a supply of palmitoylcarnitine in combination with carnitine induces apoptosis in HT-29 cells by increasing the mitochondrial respiration rate. Here we show that flavone-induced apoptosis is increased more than twofold in the presence of palmitoylcarnitine due to increased mitochondrial fatty acid transport and the subsequent metabolic generation of O₂^−. in mitochondria is the initiating factor for the execution of apoptosis. Received 12 August 2005; received after revision 12 October 2005; accepted 14 October 2005     

Interleukin 1 modulates collagen accumulation by rat granulation tissue cells both in vivo and in vitro

In in vivo studies 0.5 U human interleukin 1 (IL-1) was inoculated daily into a subcutaneously implanted viscose cellulose sponge. IL-1 significantly decreased the dry weight (7.8%) and the hydroxyproline content (24.2%) of granulomas. When the cultured rat granulation tissue cells were exposed to IL-1 (0.5-2.0 U/ml) their collagen production decreased to 80% of that in controls. No effect on cell proliferation was detected.     

Interleukin 1 modulates collagen accumulation by rat granulation tissue cells both in vivo and in vitro

Summary In in vivo studies 0.5 U human interleukin 1 (IL-1) was incoculated daily into a subcutaneously implanted viscose cellulose sponge. IL-1 significantly decreased the dry weight (7.8%) and the hydroxyproline content (24.2%) of granulomas. When the cultured rat granulation tissue cells were exposed to IL-1 (0.5–2.0 U/ml) their collagen production decreased to 80% of that in controls. No effect on cell proliferation was detected.     

Olfactory ensheathing cells are attracted to, and can endocytose, bacteria

Olfactory ensheathing cells (OECs) have been shown previously to express Toll-like receptors and to respond to bacteria by translocating nuclear factor-kappaB from the cytoplasm to the nucleus. In this study, we show that OECs extended significantly more pseudopodia when they were exposed to Escherichia coli than in the absence of bacteria (p=0.019). Co-immunoprecipitation showed that E. coli binding to OECs was mediated by Toll-like receptor 4. Lyso-Tracker, a fluorescent probe that accumulates selectively in lysosomes, and staining for type 1 lysosome-associated membrane proteins demonstrated that endocytosed FITC-conjugated E. coli were translocated to lysosomes. They appeared to be subsequently broken down, as shown by transmission electron microscopy. No obvious adherence to the membrane and less phagocytosis was observed when OECs were incubated with inert fluorescent microspheres. The ability of OECs to endocytose bacteria supports the notion that OECs play an innate immune function by protecting olfactory tissues from bacterial infection.     

Shear-dependence of endothelial functions

Endothelial cells are subjected to shear forces which influence important cell functions. Shear stress induces cell elongation and formation of stress fibers, increases permeability, pinocytosis and lipoprotein internalization, is involved in the formation of atherosclerotic lesions, increases the production of tissue plasminogen activator, and enhances von Willebrand factor release and hence platelet aggregation. It decreases adherence of erythrocytes and leukocytes, and increases the release of prostacyclin, endothelium derived relaxing factor, histamine and other compounds, but decreases erythropoietin secretion. The mechanism of signal transduction to the endothelial cell is not known exactly; shear-sensitive ion channels seem to be involved. It is concluded that a better understanding of shear-dependent endothelial functions will influence pathophysiologic concepts and therapeutic interventions.     

Induction of lymphokine-activated killer cells from nude mouse spleen cells by interleukin-4

The induction of lymphokine-activated killer (LAK) cells from natural killer (NK) lineage cells by interleukin-4 (IL-4) was studied in vitro. Activation of nude mouse spleen cells by IL-4 generated cytotoxic cells, capable of killing NK-sensitive as well as NK-resistant tumor cells. The induction of peak lytic activity was demonstrated after 3 days of culture with IL-4. Surface marker analysis indicated that the majority of precursor cells were aGM1⁺, Thy1^–, and the majority of effector cells were aGM1⁺, Thy1⁺, suggesting that IL-4 induced LAK cells from nude mouse spleen cells were similar to those from normal mouse spleen cells. The induction of nude mouse LAK cells by IL-4 was partially inhibited by anti-IL-4 or anti-interferon (IFN)-, antibody, and it was further inhibited by the combination of two antibodies, suggesting that IFN-, production was associated with LAK induction of NK lineage cells by IL-4.     

Morphological changes to Escherichia coli O157:H7, commensal E. coli and Salmonella spp in response to marginal growth conditions,with special reference to mildly stressing temperatures

Certain rod-shaped bacteria have been reported to form elongated filamentous cells when exposed to marginal growth conditions, including refrigeration temperatures. To expand upon these observations, the filamentation of commensal Escherichia coli, E. coli O157:H7 and Salmonella spp was investigated, following exposure to certain, mildly stressing, levels of temperature, pH or water activity (aw), with levels of cellular protein being monitored during cell elongation, in some experiments. Our studies indicated that cellular filamentation could be demonstrated in all 15 strains of the above organisms tested, following exposure to marginal conditions achieved by incubation at high or low temperatures, high or low pH values and low aw. The level of environmental stress causing filamentation tended to be specific to the particular organisms. For example, Salmonella spp formed filamentous cells at 44 degrees C, whereas E. coli strains, including O157, grew by binary fission at that temperature, but formed filamentous cells at 46 degrees C. In addition, plate count techniques to enumerate bacteria during filamentation, failed to reflect the increase in cell biomass that was occurring, whereas measurements of protein concentration demonstrated the increase quite strikingly. These findings have important implications for our understanding of the ability of food-borne pathogens to cause disease, since the infectious dose of a microorganism implicated in an outbreak of such disease is typically determined by a viable count method, which could underestimate the number of potential infectious units present in a food that had been stored in such a way as to provide marginal growth conditions.     

Roles for interleukin-2 (IL-2) and IL-4 in the generation of allocytotoxic T cells in the primary and secondary responses in vitro

Roles for interleukin-2(IL-2) and IL-4 in the generation of murine allocytotoxine T lymphocytes (allo-CTL) in the primary and secondary responses were studied in vitro. The generation of allo-CTL in the primary response was inhibited by anti-IL-2 monoclonal antibody (mAb), but was not inhibited by anti-IL-4 mAb. On the other hand, the generation of allo-CTL in the secondary response was partially inhibited by either anti-IL-2 or anti-IL-4 mAb, and it was almost completely inhibited by the combination of two mAbs. CD8⁺ cell-depleted splenocytes produced IL-2, but not IL-4, in response to alloantigens in the primary response, and these cells produced both IL-2 and IL-4 in the secondary response. Both exogenous IL-2 and IL-4 induced functionally active allo-CTL in the primary response from CD4⁺ cell-depleted splenocytes when these cells were stimulated with T cell-depleted allogeneic cells. These results suggest that the allo-CTL induction in the primary response is IL:-2-dependent and secondary allo-CTL induction is both IL-2 and IL-4-dependent, because unprimed CD4⁺ T cells produce IL-2, but not IL-4, whereas primed cells produce both IL-2 and IL-4 in response to alloantigens.     

Interleukin-17 stimulates inducible nitric oxide synthase-dependent toxicity in mouse beta cells

The influence of the proinflammatory cytokine interleukin (IL)-17 on inducible nitric oxide (NO) synthase (iNOS)-mediated NO release was investigated in the mouse insulinoma cell line MIN6 and mouse pancreatic islets. IL-17 markedly augmented iNOS mRNA/protein expression and subsequent NO production induced in MIN6 cells or pancreatic islets by different combinations of interferon-γ, tumor necrosis factor-α, and IL-1β. The induction of iNOS by IL-17 was preceded by phosphorylation of p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK activation completely abolished IL-17-stimulated NO release. IL-17 enhanced the NO-dependent toxicity of proinflammatory cytokines toward MIN6 cells, while IL-17-specific neutralizing antibody partially reduced the NO production and rescued insulinoma cells and pancreatic islets from NO-dependent damage induced by activated T cells. Finally, a significant increase in blood IL-17 levels was observed in a multiple low-dose streptozotocin model of diabetes, suggesting that T cell-derived IL-17 might be involved in NO-dependent damage of beta cells in this disease. Received 14 June 2005; received after revision 17 September 2005; accepted 21 September 2005     

Immunological demonstration of large quantities of glycogen or of a glycogen-like substance in human embryonic colon cells and in colon carcinomas by means of rabbit antisera raised against a strain of Escherichia coli 013]

Rabbit antisera raised against a strain of E. coli 013, with a strong antiglycogen activity, were tested on human fetal and normal adult colons, on colon carcinomas, and on colon tumor cells in culture (HT29). Only very rare granules were present in adult normal colons when tested with the immunofluorescence method. In faetal colons, in 12 out of 14 carcinomas, and on HT29 cells, the immunofluorescent reactions were similar to those observed in normal liver. The reactions were negative after previous treatment with alpha-amylase. They were inhibited with glycogen, with phenol-alcohol, perchloric, and trichloroacetic extracts from faetal colons, and with a tumor trichloroacetic extract. The extracts precipitated with anti-E. coli 013 antisera. They had a strong inhibiting activity in a radioimmunoassay test with labeled glycogen. The extracts from normal adult colons did not precipitate with the antisera and they had no inhibiting activity in either immunofluorescence and radioimmunoassay tests.     

Roles for interleukin-2 (IL-2) and IL-4 in the generation of allocytotoxic T cells in the primary and secondary responses in vitro.

Roles for interleukin-2 (IL-2) and IL-4 in the generation of murine allocytotoxine T lymphocytes (allo-CTL) in the primary and secondary responses were studied in vitro. The generation of allo-CTL in the primary response was inhibited by anti-IL-2 monoclonal antibody (mAb), but was not inhibited by anti-IL-4 mAb. On the other hand, the generation of allo-CTL in the secondary response was partially inhibited by either anti-IL-2 or anti-IL-4 mAb, and it was almost completely inhibited by the combination of two mAbs. CD8+ cell-depleted splenocytes produced IL-2, but not IL-4, in response to alloantigens in the primary response, and these cells produced both IL-2 and IL-4 in the secondary response. Both exogenous IL-2 and IL-4 induced functionally active allo-CTL in the primary response from CD4+ cell-depleted splenocytes when these cells were stimulated with T cell-depleted allogeneic cells. These results suggest that the allo-CTL induction in the primary response is IL:-2-dependent and secondary allo-CTL induction is both IL-2 and IL-4-dependent, because unprimed CD4+ T cells produce IL-2, but not IL-4, whereas primed cells produce both IL-2 and IL-4 in response to alloantigens.     

Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages

Enterococci are commensal organisms in the alimentary tract. However, they can cause a variety of life-threatening infections, especially in nosocomial settings. We hypothesized that induction of cell death might enable these facultative pathogenic bacteria to evade the innate immune response and to cause infections of their host. We demonstrate that E. faecium when exposed to lysozyme induces cell death in macrophages in vitro and in vivo. Flow cytometric analyses of J774A.1 macrophages infected with E. faecium revealed loss of cell membrane integrity indicated by uptake of propidium iodide and decrease of the inner mitochondrial transmembrane potential ΔΨ_m. Inhibition of caspases, treatment of macrophages with cytochalasin D, or rifampicin did not prevent cells from dying, suggesting cell death mechanisms that are independent of caspase activation, bacterial uptake, and intracellular bacterial replication. Characteristics of necrotic cell death were demonstrated by both lack of procaspase 3 activation and cell shrinkage, electron microscopy, and release of lactate dehydrogenase. Pretreatment of E. faecium with lysozyme and subsequently with broad spectrum protease considerably reduced cell death, suggesting that a bacterial surface protein is causative for cell death induction. Moreover, in a mouse peritonitis model we demonstrated that E. faecium induces cell death of peritoneal macrophages in vivo. Altogether, our results show that enterococci, under specific conditions such as exposure to lysozyme, induce necrotic cell death in macrophages, which might contribute to disseminated infections by these facultative pathogenic bacteria.     

Agonist-induced internalization and desensitization of the human nociceptin receptor expressed in CHO cells

In this study, we examined agonist-induced internalization, recycling and signalling (measure of cAMP levels) of the cloned human nociceptin receptor (hNOP) expressed in CHO-K1 cells. Internalization was proven by a receptor-binding assay on viable cells. The agonist nociceptin/orphanin FQ (NC) promoted rapid internalization of the hNOP receptor (approximately 78% of cell surface receptors were lost after 2 min exposure to 1 microM NC) in a clathrin- and ATP-dependent manner. Internalization was more rapid and marked in CHO-K1 cells than, as we previously reported, in SK-N-BE cells. This difference may be related to higher levels of beta-arrestin isoforms detected in CHO-K1 than in SK-N-BE cells. hNOP receptor internalization was partially reversible and recycling occurred in the presence of the agonist; receptor recycling was dependent on okadaic acid-sensitive phosphatases and was blocked by monensin. Confocal microscopy analysis confirmed the internalization and the recycling back to the plasma membrane of an epitope-tagged hNOP receptor expressed in CHO-K1 cells. These receptors underwent rapid desensitization upon agonist challenge: NC efficacy in inhibiting forskolin-stimulated cAMP production was significantly reduced 10 min after exposure and correlated with the rate of receptor internalization. Moreover, we observed that blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. Thus, the dynamic cycle between hNOP receptor activation, internalization and recycling determines the activity of this receptor on the cell surface.     

An MD2-derived peptide promotes LPS aggregation,facilitates its internalization in THP-1 cells,and inhibits LPS-induced pro-inflammatory responses

MD2, a 160-residue accessory glycoprotein, is responsible for the recognition and binding of Gram-negative bacterial membrane component, lipopolysaccharide (LPS). Internalization of pathogen inside the mononuclear phagocytes has also been attributed to MD2 which leads to the clearance of pathogens from the host. However, not much is known about the segments in MD2 that are responsible for LPS interaction or internalization of pathogen inside the defense cells. A 16-residue stretch (MD54) from MD2 protein has been identified that possesses a short heptad repeat sequence and four cationic residues enabling it to participate in both hydrophobic and electrostatic interactions with LPS. An MD54 analog of the same size was also designed in which a leucine residue at a heptadic position was replaced with an alanine residue. MD54 but not its analog, MMD54 induced aggregation of LPS and aided in its internalization within THP-1 monocytes. Furthermore, MD54 inhibited LPS-induced nuclear translocation of NF-κB in PMA-treated THP-1 and TLR4/MD2/CD14-transfected HEK-293T cells and the production of pro-inflammatory cytokines. In addition, in in vivo experiments, MD54 showed marked protection and survival of mice against LPS-induced inflammation and death. Overall, we have identified a short peptide with heptad repeat sequence from MD2 that can cause aggregation of LPS and abet in its internalization within THP-1 cells, resulting in attenuation of LPS-induced pro-inflammatory responses in vitro and in vivo.