Prostaglandin production by amnion and decidual cells in response to bacterial products

Media from bacterial cultures have been tested for actions on prostaglandin biosynthesis by human amnion and decidual cells. The bacterial species, which are commonly associated with intrauterine infections, were Group B streptococcus, Escherichia coli, Fusobacterium nucleatum, Bacteroides fragilis, Gardnerella vaginalis, Neisseria gonorrhea, Mycoplasma hominis and Ureaplasma urealyticum. Overall, low doses of bacterial products were stimulatory of amnion prostaglandin production, whereas high doses were inhibitory. A similar pattern of results was obtained for effects on decidual prostaglandin production, although stimulatory actions at low doses were less pronounced. In all experiments interleukin 1 beta consistently induced a stimulation of prostaglandin production that greatly exceeded that caused by any bacterial product. It is possible that the inhibitory action of high doses of bacterial products on prostaglandin biosynthesis may contribute to the poor course of labor experienced by women with chorioamnionitis. Furthermore, these data lend credence to the view that the host response to infection (i.e. cytokine secretion) is the major mediator of subsequent preterm labor.     

Regulation of proteinase-activated receptor 1 by inflammatory mediators in human vascular endothelial cells

Thrombin plays a critical role in haemostasis, inflammation, and cell proliferation, mediated by proteinase-activated receptor 1 (PAR-1; thrombin receptor). The physiological and pathological regulation of PAR-1 by inflammatory mediators has not yet been fully elucidated. The aim of this study is to investigate the effects of inflammatory mediators on mRNA and protein expression of PAR-1 in early passage human vascular endothelial cells. Endothelial cells were activated by inflammatory mediators, such as tumour necrosis factor alpha (TNFalpha), interferon gamma (IFN gamma), and bacterial substance lipopolysaccharide (LPS), and the PAR-1 expression was verified by flow cytometry or RT-PCR. By stimulating endothelial cells with TNFalpha, IFN gamma, and LPS, the PAR-1 expression on the cell surface remained almost unchanged for 48 h. After stimulation with 20-300 U/ml TNFalpha, the total cellular PAR-1 expression (both on cell surface and in the cytoplasm) significantly decreased at 24h and thereafter recovered to the basal level at 48 h. The stimulation with 100 U/ml TNFalpha transiently down-regulated the PAR-1 mRNA expression to approximately 0.3-fold of the basal level at 30 min, but it rebounded 3-fold above the basal level at 6h, and again decreased to 0.5-fold of the basal level at 12h, and finally returned to the basal level at 24h. In contrast, IFN gamma or LPS did not affect the PAR-1 mRNA expression.     

Prostaglandin and thromboxane production by rat decidual microsomes

The preparation of a microsomal fraction from the decidual tissue of pregnant rat uteri is described. Incubation of such microsomes with a mixture of radiolabelled and cold arachidonic acid (51 micrometer) plus cofactors resulted in a 30% substrate conversion. Products were resolved into four peaks (A, B, C and D) by thin-layer chromatography. Combined gas-liquid chromatography-mass spectrometry and further thin-layer chromatography identified the products as PGF2 alpha (A); thromboxane B2 (B); a mixture of 6-OXO PGF1 alpha and PGE2 (C); PGD2 and PGE2 (D). PGE2 was the major product.     

Bacterial lipopolysaccharide and inflammatory mediators augment IL-6 secretion by human endothelial cells

The interaction between human endothelial cells and leukocytes during immunologic and inflammatory responses is in part mediated through the release of soluble mediators. We report that cultured human umbilical vein endothelial cells secrete IL-6 when stimulated with LPS. This effect was inhibited by polymyxin-B. The monokines IL-1 and TNF-alpha were also potent inducers of IL-6, whereas lymphotoxin was only effective at much higher concentrations. Endothelial cell supernatant IL-6 was active as hybridoma-plasmacytoma growth factor and as B-cell stimulating factor. Endothelial IL-6 activity was neutralized by a specific anti-IL-6 antibody and by immunoprecipitation it was shown to be identical in size to human fibroblast-derived IL-6. As IL-6 is possibly an important regulator of host defense responses, production of this cytokine by endothelial cells may contribute to the pathogenesis of various inflammatory and immunologic diseases.     

Statins inhibit in vitro calcification of human vascular smooth muscle cells induced by inflammatory mediators

Although lipid-lowering therapy with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) decreases the progression of coronary artery and aortic valve calcification, the mechanism of action of these drugs to inhibit the calcification process remains unclear. In this study, we investigated the effect of statins such as cerivastatin and atorvastatin on vascular calcification by utilizing an in vitro model of inflammatory vascular calcification. Cerivastatin and atorvastatin dose-dependently inhibited in vitro calcification of human vascular smooth muscle cells (HVSMCs) induced by the following inflammatory mediators (IM): interferon-gamma, 1alpha,25-dihydroxyvitamin D3, tumor necrosis factor-alpha, and oncostatin M. These statins also depressed expression of alkaline phosphatase (ALP) in HVSMCs induced by these factors. Mevalonate and geranylgeranylpyrophosphate reversed the inhibitory effect of cerivastatin on ALP expression in HVSMCs, while farnesylpyrophosphate showed no effect on the ALP activities inhibited by this drug, suggesting that inhibition of Rho and its downstream target, Rho kinase may mediate the inhibitory effect of cerivastatin. Cerivastatin prevented RhoA activation in HVSMCs induced by the IM. A specific inhibitor of Rho kinase (Y-27632) inhibited in vitro calcification and induction of ALP in HVSMCs. These findings provide a possible mechanism of statins to prevent the progression of calcification in inflammatory vascular diseases such as atherosclerosis and cardiac valvular calcification.     

Estrogen metabolites in the release of inflammatory mediators from human amnion-derived cells

AimsHuman amnion-derived cells have been used as in vitro models to test the release of inflammatory mediators, such as arachidonic acid (AA) and prostaglandin E₂ (PGE₂). We compared estrogen metabolites for their ability to induce AA release, to influence PGE₂ production and to interact toward intracellular estrogen receptors (ERs).Main methodsMetabolite effects on AA and PGE₂ release were examined by radiolabelled substrate incorporation and by colorimetric enzyme immunoassays, respectively. [³H]17-β-estradiol binding displacements were performed on Ro-20-1724 treated whole cells.Key findingsIn WISH cells, estrone, 2-hydroxy-estrone and estriol induced a rapid dose dependent release of AA that was not inhibited by cycloheximide. Estrone and 2-hydroxy-estrone showed biphasic dose–response curves of PGE₂, whereas estriol and 16-α-hydroxy-estrone increased PGE₂ levels at high concentrations. 2-methoxy-estrone, 4-hydroxy-estradiol and 4-hydroxy-estrone did not significantly affect PGE₂ release. 2-methoxy-estradiol and 2-hydroxy-estradiol decreased the PGE₂ release. Effects of metabolites on PGE₂ were inhibited by cycloheximide and by the ER antagonist tamoxifen. In AV3 cells PGE₂ production was poorly detectable. On Ro-20-1724 treated WISH cells the K_i of 17-β-estradiol was 29.2 ± 5.4 nM. Estrone, 2-methoxy-estrone and 2-methoxy-estradiol showed similar affinity values. The hydroxyl substituent at position 2, 4 and 16 decreased or markedly increased the affinity for estradiol or estrone derivatives, respectively.SignificanceThe estrogen metabolites induced nongenomic effects on AA release from WISH cells. The influence on PGE₂ release was detectable only on WISH cells. These effects appeared genomic and mediated by intracellular ERs, whose properties seemed strongly dependent on intracellular cAMP levels.     

Secretion of inflammatory mediators by isolated rat Kupffer cells: the effect of octreotide

Aims: We studied the production of inflammatory mediators by rat KC and the possible in vitro effect of the somatostatin analogue octreotide. Methods: Primary KC cultures were incubated with LPS added alone or with different concentrations of octreotide. The production of TNF, IL-6, IL-10, IL-12 and IL-13 was assessed in culture supernatants by ELISA and that of nitric oxide (NO) by a modification of the Griess reaction. Results: Isolated KC produced a basal amount of TNF, IL-6, IL-12, IL-13, and NO but not IL-10. LPS-stimulated KC secreted significantly increased amounts of TNF (P<0.001), IL-6 (P<0.01), IL-10 (P<0.001), IL-12 (P<0.01), and NO (P<0.001) whereas IL-13 production remained constant. Octreotide reduced IL-12 (P<0.05) and increased IL-13 (P<0.05) production by unstimulated KC. Furthermore, octreotide suppressed TNF production (P<0.05), without modifying TNF mRNA expression and decreased iNOS expression and NO (P≈0.05) production by LPS-activated KC. These effects were reversed with Wortmannin pre-treatment suggesting that octreotide may act via interference with phosphatidylinositol 3-kinase pathways. Conclusions: These data demonstrate that KC is a source of multiple inflammatory mediators, indicating a critical role in liver inflammatory disorders. Octreotide modulates inflammatory mediator production by isolated KC, suggesting that it might have immunoregulatory and anti-inflammatory effects in liver diseases.     

Prostaglandin synthesis by human glomerular cells in culture

PG synthesis by cultured human glomerular mesangial and epithelial cells incubated with [1- 14C] arachidonic acid was determined by radioimmunoassay (RIA) after high performance liquid chromatography purification. Both dissociated cells and cell monolayers were studied under basal conditions. PG synthesis by epithelial cells was undetectable. Mesangial cells produced low amounts of PGE2, PGF2 alpha and 6 keto-PGF1 alpha and no TXB2. We also examined the effects of several agents on PG synthesis in these two types of cells scraped away from their flasks using direct RIA. Arachidonic acid produced a slight stimulation only with mesangial cells whereas angiotensin II, cyclic AMP and calcium ionophore were inactive with both cell lines. Homogenization of the cells did not enhance the stimulatory effect of arachidonic acid. Alkalinization of the incubation medium produced an increase of PG production by mesangial cells. These results suggest that two types of human glomerular cells, particularly epithelial cells, possess low cyclooxygenase activity. The low capacity of human mesangial and epithelial cells to produce PG may have consequences for the endocrine control of the glomerular microcirculation in man.     

Prostaglandin secretion by human thymic epithelium: In vitro effects of steroids

Increasing evidence suggests a role for prostaglandins in the immune response. As steroids have been shown recently to modulate prostaglandin secretion, we have studied the secretion of prostaglandin and the effect of various steroids in culture of human thymus epithelial cells. Using reverse phase high pressure liquid chromatography and radioimmunoassays, we have shown that these cells produce substantial amounts of PGE₂ and PGE_2α and that this secretion is modulated by steroid hormones. Prostaglandin could represent one of the factors of the thymic environment which respond to steroid hormones.     

Hematopoiesis sculpted by pathogens: Toll-like receptors and inflammatory mediators directly activate stem cells

Hematopoietic stem cells (HSCs) repopulate the immune system during normal replenishment as well as under the burden of pathogen stress, but the respective outcomes of differentiation are not the same. Under homeostatic conditions such as those which accompany turnover of immune cell subsets, HSCs appear to co-equally prime genes associated with the major downstream lineages: lymphoid, myeloid, and megakaryocyte/erythroid. Recent studies reveal, however, that during pathogen exposure, hematopoiesis may yield progeny in proportions different than those produced under homeostasis. At least some of these effects may be due to pathogen engagement of Toll-like receptors (TLRs) expressed on HSCs. HSCs are also responsive to inflammatory cytokines that are produced in response to pathogen burden and are present in the bone marrow microenvironment. Thus, hematopoiesis is not a formulaic process that produces the same, predictable outcome regardless of the specific environmental context. Rather, hematopoiesis represents a dynamic biological system that can be appreciably responsive to environmental factors, an influence that extends to the level of the HSC itself. Knowledge of functional consequences of TLR ligation on HSCs may be therapeutically exploited and applied to treatment of hematopoietic insufficiency in the setting of infection and disease.     

Prostaglandin synthesis in decidual tissue of the rat uterus

Prostaglandin (PG) synthetase activity and tissue concentration were measured in unilateral deciduomata induced by traumatization of the pseudopregnant rat uterus and in the decidua of pregnancy. PG synthetase activity per unit weight of deciduoma tissue was 7–10 fold higher, throughout the life-span of the deciduoma, than that in the untraumatized control horn. The concentration of prostaglandins of the E-type in the deciduoma exceeded that found in the control uterine horn by a factor of 10–20 on days 3–4 after decidual induction, and about five-fold on days 9–10. The concentration of prostaglandins of the F-type in the deciduoma measured on days 4 and 8 did not differ significantly from that in the control horn.

In the decidua of pregnant rats, both PG synthetase activity and PGE content were 20–40 times higher than the corresponding values for the myometrium of the same horn. The physiological role of the high level of prostaglandin production in decidual tissue requires further investigation.     

Chemokines: inflammatory mediators of atherosclerosis

Atherosclerosis as the underlying mechanisms of myocardial infarction, stroke and peripheral artery disease remains the major cause of morbidity and mortality in developed countries. Recent developments in vascular biology have indicated that atherosclerosis can be best characterized as a chronic inflammatory disease of the vessel wall that promotes lesion development and progression. Chemokines regulate and control these processes by orchestrating adhesive interactions of circulating blood cells with the arterial wall and their subsequent extravasation. Exhibiting a high degree of specialization and cooperation, different chemokines mediate distinct steps during the atherogenic recruitment of monocytes and T cells. This diversity of chemokine expression and function might lead to the identification of selective therapeutic targets for the prevention and treatment of atherosclerosis.     

B cells as under-appreciated mediators of non-auto-immune inflammatory disease

B lymphocytes play roles in many auto-immune diseases characterized by unresolved inflammation, and B cell ablation is proving to be a relatively safe, effective treatment for such diseases. B cells function, in part, as important sources of regulatory cytokines in auto-immune disease, but B cell cytokines also play roles in other non-auto-immune inflammatory diseases. B cell ablation may therefore benefit inflammatory disease patients in addition to its demonstrated efficacy in auto-immune disease. Current ablation drugs clear both pro- and anti-inflammatory B cell subsets, which may unexpectedly exacerbate some pathologies. This possibility argues that a more thorough understanding of B cell function in human inflammatory disease is required to safely harness the clinical promise of B cell ablation. Type 2 diabetes (T2D) and periodontal disease (PD) are two inflammatory diseases characterized by little autoimmunity. These diseases are linked by coincident presentation and alterations in toll-like receptor (TLR)-dependent B cell cytokine production, which may identify B cell ablation as a new therapy for co-affected individuals. Further analysis of the role B cells and B cell cytokines play in T2D, PD and other inflammatory diseases is required to justify testing B cell depletion therapies on a broader range of patients.     

Prostaglandin biosynthesis by lapine articular chondrocytes in culture

Secondary monolayer and spinner cultures of rabbit articular chondrocytes released into the culture medium prostaglandins the synthesis of which was inhibited by sodium meclofenamate. The prostaglandins measured by radioimmunoassay were, in order of decreasing abundance, prostaglandin E₂, 6-oxoprostaglandin F_1α, (the stable metabolite of prostacyclin) and prostaglandin F_2α. Several lines of evidence indicated that chondrocytes synthesize little if any thromboxane B₂ (the stable metabolite of thromboxane A₂). The presence of prostaglandins was confirmed by radiometric thin-layer chromatography of extracts of culture media incubated with [³H]arachidonic acid-labeled cells. In monolayer culture, chondrocytes synthesized immunoreactive prostaglandins in serum-free as well as serum-containing medium. Monolayer chondrocytes produced higher levels of prostaglandin E₂ relative to 6-oxo-prostaglandin F_1α than did spinner cells, but the latter synthesized more total prostaglandins. The identity of endogenous prostaglandins as well as those synthesized in short-term culture by rabbit cartilage slices was compared to those produced by chondrocytes in long-term culture. Chondrocytes synthesized all of the prosta-glandins found in articular cartilage. Minimal quantities of thromboxane B₂ were detected in cartilage. A higher percentage of 6-oxo-prostaglandin F_1α relative to other prostaglandins was found in cartilage than in either monolayer or spinner chondrocyte cultures. These results demonstrate that articular chondrocytes synthesize prostaglandins and prostacyclin. These prostaglandins may exert significant physiological effects on cartilage, since exogenous prosta-glandins depress chondrocyte sulfated-proteoglycan synthesis and may even promote proteoglycan degradation.     

Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-β (IFN-β) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-β. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-κB and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88) MyD88 expression and NF-κB activation, confirming that hypoxia suppressed the LPS-induced inflammatory response by affecting TLR4 signaling. In conclusion, our results demonstrated that hypoxia attenuated the host immune and inflammatory response against Acanthamoeba infection by suppressing TLR4 signaling, indicating that hypoxia might impair the host cell's ability to eliminate the Acanthamoeba invasion and that hypoxia could enhance cell susceptibility to Acanthamoeba infection. These results may explain why contact lens use is one of the most prominent risk factors for AK.