Effects of sphondin,isolated from Heracleum laciniatum,on IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells

Recently, under large-scale screening experiments, we found that sphondin, a furanocoumarin derivative isolated from Heracleum laciniatum, possessed an inhibitory effect on IL-1beta-induced increase in the level of COX-2 protein and PGE(2) release in A549 cells. Accordingly, we examined in the present study the action mechanism of sphondin on the inhibition of IL-1beta-induced COX-2 protein expression and PGE(2) release in a human pulmonary epithelial cell line (A549). Pretreatment of cells with sphondin (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 protein expression and PGE(2) release. The IL-1beta-induced increase in COX-2 mRNA expression was also attenuated by sphondin (50 microM). The selective COX-2 inhibitor, NS-398 (0.01-1 microM), inhibited the activity of the COX-2 enzyme in a concentration-dependent manner, while sphondin (10-50 microM) had no effect. Sphondin (50 microM) did not affect the IL-1beta-induced activations of p44/42 MAPK, p38 MAPK, and JNK. Treatment of cells with sphondin (50 microM) or the NF-kappaB inhibitor, PDTC (50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol and translocation of p65 NF-kappaB from the cytosol to the nucleus. Furthermore, IL-1beta-induced NF-kappaB-specific DNA-protein complex formation in the nucleus was partially inhibited by sphondin (50 microM) or PDTC (50 microM). Taken together, we demonstrate that sphondin inhibits IL-1beta-induced PGE(2) release in A549 cells; this inhibition is mediated by suppressing of COX-2 expression, rather than by inhibiting COX-2 enzyme activity. The inhibitory mechanism of sphondin on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. We conclude that sphondin may have the therapeutic potential as an anti-inflammatory drug on airway inflammation.     

The effect of dehydroepiandrosterone on inflammatory response of astroglial cells

An increased interest in neuroinflammation is conditioned by its involvement in various pathological processes in the brain. Astrocytes play an important role in neuroinflammation, participating in its regulation, throwing out a large number of signaling molecules. Steroid compounds, actively produced by astrocytes, are of interest with regards to the regulation of inflammatory processes in the central nervous system. In the present work the effect of dehydroepiandrosterone (DHEA) on astroglial cells (cultured primary rat astrocytes) in a model of inflammation was studied. The inflammatory response was stimulated with lipopolysaccharide (LPS). Expression levels of pro-inflammatory factor TNFα, antinflammatory interleukin IL-10, and both pro- and antiinflammatory protein COX-2 were measured. The expression of IL-10, COX-2, and TNFα mRNA was determined by real-time PCR, COX-2 protein level by immunoblotting method, TNFα and IL-10 release by enzyme immunoassay. The effect of short-term (30 min) and long-term (24 h) exposure to DHEA was evaluated. It was shown that DHEA potentiates LPS-stimulated (1) increase in the IL-10 mRNA level; (2) IL-10 release; (3) does not affect TNFα level, and (4) exerts a weak pulsating bidirectional effect on COX-2. Using trilostane, an inhibitor of 3β-hydroxysteroid dehydrogenase, a key enzyme of DHEA metabolism, it was shown that DHEA metabolites make the main contribution to its effect. Thus, DHEA is of interest as a stimulant of anti-inflammatory processes in the brain.     

Zinc modulates mRNA levels of cytokines

Zinc plays an important role in cell-mediated immune function. Altered cellular immune response resulting from zinc deficiency leads to frequent microbial infections, thymic atrophy, decreased natural killer activity, decreased thymic hormone activity, and altered cytokine production. In this study, we examined the effect of zinc deficiency on IL-2 and IFN-gamma in HUT-78 (Th0) and D1.1 (Th1) cell lines and TNF-alpha, IL-1 beta, and IL-8 in the HL-60 (monocyte-macrophage) cell line. The results demonstrate that zinc deficiency decreased the levels of IL-2 and IFN-gamma cytokines and mRNAs in HUT-78 after 6 h of PMA/p-phytohemagglutinin (PHA) stimulation and in D1.1 cells after 6 h of PHA/ionomycin stimulation compared with the zinc-sufficient cells. However, zinc deficiency increased the levels of TNF-alpha, IL-1 beta, and IL-8 cytokines and mRNAs in HL-60 cells after 6 h of PMA stimulation compared with zinc-sufficient cells. Actinomycin D study suggests that the changes in the levels of these cytokine mRNAs were not the result of the stability affected by zinc but might be the result of altered expression of these cytokine genes. These data demonstrate that zinc mediates positively the gene expression of IL-2 and IFN-gamma in the Th1 cell line and negatively TNF-alpha, IL-1 beta, and IL-8 in the monocyte-macrophage cell line. Our study shows that the effect of zinc on gene expression and production of cytokines is cell lineage specific.     

Induction of a CD4+ T regulatory type 1 response by cyclooxygenase-2-overexpressing glioma

PGE(2), synthesized by cyclooxygenase-2 (COX-2)-overexpressing tumor, is known to contribute to cellular immune suppression in cancer patients, but the mechanism remains unclear. We report the mechanism of a CD4(+) T regulatory type 1 (Tr1) induction by CD11c(+) mature dendritic cells (DCs) that phagocytose allogeneic and autologous COX-2-overexpressing glioma. A human glioma cell line, U-87MG, and primary cultured glioblastoma cells (MG-377) overexpressed COX-2. We did not detect IL-10Ralpha expression in these gliomas, and rIL-10 did not suppress their COX-2 expression. Exposure to COX-2-overexpressing glioma induced mature DCs to overexpress IL-10 and decreased IL-12p70 production. These DCs induced a Tr1 response, which is characterized by robust secretion of IL-10 and TGF-beta with negligible IL-4 secretion by CD4(+) T cells, and an inhibitory effect on admixed lymphocytes. Peripheral CD4(+) T cell populations isolated from an MG-377 patient also predominantly demonstrated a Tr1 response against MG-377 cells. Selective COX-2 inhibition in COX-2-overexpressing gliomas at the time of phagocytic uptake by DCs abrogated this regulatory response and instead elicited Th1 activity. COX-2 stable transfectants in LN-18 (LN-18-COX2) also induced a Tr1 response. The effect of a COX-2 inhibition in LN-18-COX2 is reversible after administration of PGE(2). Taken together, robust levels of PGE(2) from COX-2-overexpressing glioma, which is unresponsive to IL-10 within the local microenvironment, may cause DCs to secrete high levels of IL-10. These results indicate that COX-2-overexpressing tumors induce a Tr1 response, which is mediated by tumor-exposed, IL-10-enhanced DCs.     

Transcriptional regulation of cyclooxygenase-2 in the Human Microvascular Endothelial Cell Line,HMEC-1: Control by the combinatorial actions of AP2, NF-IL-6 and CRE elements

Interleukin-1beta (IL-1) is a potent inducer of cyclooxygenase-2 (COX-2) and prostaglandin biosynthesis in many types of cells, yet little is known about the molecular mechanisms regulating IL-1 mediated prostanoid biosynthesis in the endothelium of the microvasculature. Therefore, we examined the cis- and trans-acting factors regulating IL-1-induced COX-2 expression in the human microvascular endothelial cell line, HMEC-1. IL-1 enhanced steady state levels of COX-2 protein and mRNA synthesis by approximately 2-fold which preceded a 2-fold increase in PGF(alpha) biosynthesis. Expression of a series of COX-2 promoter-luciferase constructs in IL-1 treated HMEC-1 cells revealed that the 'full length' (-1432/+59 bp) promoter was 10 times more active than the SV-40 promoter/enhancer and that it could be further activated by IL-1. Surprisingly however, all except for the shortest COX-2 promoter construct retained the ability to respond to IL-1 and luciferase activity driven by -191/+59 bp COX-2 promoter was as responsive to IL-1 as the full-length promoter. Moreover, site-directed promoter mutagenesis and electophoretic mobility shift assays (EMSA) indicate that the combinatorial actions of AP2, NF-IL6, and CRE elements are critical for both constitutive and IL-1-inducible COX-2 promoter activity. Understanding the mechanism(s) regulating COX-2 gene expression and prostaglandin biosynthesis in the microvasculature has important implications with regard to inflammation and angiogenesis in vivo.     

Prostaglandin E(2) stimulates prostatic intraepithelial neoplasia cell growth through activation of the interleukin-6/GP130/STAT-3 signaling pathway.

Cyclooxygenase (COX)-2 expression and prostaglandin E(2) (PGE(2)) secretion are increased in prostatic intraepithelial neoplasia (PIN) and prostate cancer. PGE(2) biosynthesis by cyclooxygenase (COX)-2 plays a pivotal role in inflammation and carcinogenesis. One of the critical proinflammatory cytokines in the prostate is interleukin-6 (IL-6). We hypothesized that increased expression of COX-2, with resultant increased levels of PGE(2) in human PIN cells, activates the IL-6 signaling pathway. We demonstrate an autocrine upregulation of PGE(2) mediated by IL-6 in a human PIN cell line. We further demonstrate that PGE(2) stimulates soluble IL-6 receptor (sIL-6R) release, gp130 dimerization, Stat-3 protein phosphorylation, and DNA binding activity. These events, induced by PGE(2), lead to increased PIN cell growth. Treatment of PIN cells with a selective COX-2 inhibitor decreases cell growth. Finally, PGE(2)-stimulated PIN cell growth was abrogated by the addition of IL-6 neutralizing antibodies. These data provide mechanistic evidence that increased expression of COX-2/PGE(2) contributes to prostate cancer development and progression via activation of the IL-6 signaling pathway.     

Nuclear factor kappa B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1beta

Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin (PG) output by up-regulating the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG synthesis. In this study, we investigated the possible role of the nuclear factor kappa B (NFkappaB) in IL-1beta signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1beta, and activation of NFkappaB was assessed by measuring changes in the inhibitory protein IkappaB (total IkappaB and phosphorylated IkappaB) using Western blot analysis as well as by nuclear binding of NFkappaB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1beta, phosphorylated IkappaB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IkappaB and increased nuclear NFkappaB DNA-binding activity. In the IL-1beta-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1beta stimulation. When NFkappaB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFkappaB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFkappaB is involved in the IL-1beta-induced COX-2 expression in the mesenchymal cells of human amnion.     

Inhibition of the expression of alpha-smooth muscle actin in human hepatic stellate cell line,LI90, by a selective cyclooxygenase 2 inhibitor,NS-398

Cyclooxygenase 2 (COX-2) has been thought to be associated with liver fibrosis whereas it is well known that hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis. There is little evidence of how COX-2 regulates the activation of human HSC or the mechanism involved. In this study, we investigated the effect of a COX-2 inhibitor, NS-398, on a line of human HSC, LI90. Our findings demonstrated that alpha-smooth muscle actin (alpha-SMA) protein expression was inhibited in a dose-dependent manner by treatment with NS-398. Proliferation cell nuclear antigen (PCNA) expression and cell growth were partially down-regulated. The generation of PGE2, IL-8, IL-6, and hyaluronan in the cultured medium was also inhibited. In conclusion, our findings imply that a selective COX-2 inhibitor might be a potential drug for the chemoprevention and treatment of liver fibrosis by inhibiting the activation of HSC.     

Endothelin-1 increases expression of cyclooxygenase-2 and production of interlukin-8 in hunan pulmonary epithelial cells

Inducible cyclooxygenase (COX-2) and inflammatory cytokines play important roles in inflammatory processes of chronic obstructive pulmonary disease (COPD). Endothelin-1 (ET-1) might be also involved in the pathophysilogical processes in COPD. In the present study, we determined whether ET-1 could regulate the expression of COX-2 and alter the production of interleukin-8 (IL-8) in human pulmonary epithelial cells (A549). Induced sputum samples were collected from 13 stable COPD patients and 14 healthy subjects. The COX-2 protein, ET-1, PGE(2) and IL-8 in these sputum samples were analyzed. A549 cells were incubated with ET-1 in the presence or absence of celecoxib, a selective COX-2 inhibitor. The expression of COX-2 protein in the cell and the amounts of PGE(2) and IL-8 in the medium were measured. The levels of COX-2 protein, ET-1, PGE(2) and IL-8 were significantly increased in induced sputum from COPD patients when compared to healthy subjects. ET-1 increased the expression of COX-2 protein, as well as the production of PGE(2) in A549 cells. Increased production of PGE(2) was inhibited by celecoxib. ET-1 also increased the production of IL-8. Interestingly, ET-1-induced production of IL-8 was also inhibited by celecoxib. These findings indicate that ET-1 plays important roles in regulating COX-2 expression and production of IL-8 in A549 cells. ET-1 mediated production of IL-8 is likely through a COX-2-dependent mechanism.     

Cutting edge: cyclooxygenase-2 activation suppresses Th1 polarization in response to Helicobacter pylori

Helicobacter pylori infection causes a Th1-driven mucosal immune response. Cyclooxygenase (COX)-2 is up-regulated in lamina propria mononuclear cells in H. pylori gastritis. Because COX-2 can modulate Th1/Th2 balance, we determined whether H. pylori activates COX-2 in human PBMCs, and the effect on cytokine and proliferative responses. There was significant up-regulation of COX-2 mRNA and PGE(2) release in response to H. pylori preparations. Addition of COX-2 inhibitors or an anti-PGE(2) Ab resulted in a marked increase in H. pylori-stimulated IL-12 and IFN-gamma production, and a decrease in IL-10 levels. Addition of PGE(2) or cAMP, the second messenger activated by PGE(2), had the opposite effect. Similarly, stimulated cell proliferation was increased by COX-2 inhibitors or anti-PGE(2) Ab, and was decreased by PGE(2). Our findings indicate that COX-2 has an immunosuppressive role in H. pylori gastritis, which may protect the mucosa from severe injury, but may also contribute to the persistence of the infection.     

Differential pattern of inflammatory molecule regulation in intestinal epithelial cells stimulated with IL-1

To better predict the consequences of blocking signal transduction pathways as a means of controlling intestinal inflammation, we are characterizing the pathways up-regulated by IL-1 in intestinal epithelial cells (IEC). IL-1beta induced increased mRNA levels of MIP-2, MCP-1, RANTES, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) in the IEC-18 cell line. IL-1beta activated NF-kappaB but not ERK or p38. Infecting cells with adenovirus expressing a mutated gene for IkappaBalpha (IkappaBAA) blocked IL-1-induced mRNA increases in MIP-2, MCP-1, and iNOS but not COX-2 or RANTES. Expression of IkappaBAA attenuated the IL-1-induced increase in COX-2 protein. Unexpectedly, RANTES mRNA increased, and protein was secreted by cells expressing IkappaBAA in the absence of IL-1. Adenovirus-expressing IkappaBAA, blocking protein synthesis, and IL-1beta all resulted in activation of JNK. The JNK inhibitor SP600125 prevented the RANTES increases by all three stimuli. A human enterocyte line was similarly examined, and both NF-kappaB and JNK regulate IL-1-induced RANTES secretion. We conclude that in IEC-18, IL-1beta-induced increases in mRNA for MIP-2, MCP-1, and iNOS are NF-kappaB-dependent, whereas regulation of RANTES mRNA is independent of NF-kappaB but is positively regulated by JNK. IL-1beta-induced mRNA increases in COX-2 mRNA are both NF-kappaB- and MAPK-independent but the translation of COX-2 protein is NF-kappaB-dependent. This pattern of signaling due to a single stimulus exposed the complexities of regulating inflammatory genes in IEC.     

Expression of natural autoantibodies in MRL-lpr mice protects from lupus nephritis and improves survival

Natural autoantibodies (NAA) and their associated B cells constitute a substantial proportion of the normal Ab and B cell repertoire. They often have weak reactivity toward a variety of self-Ags such as DNA, nucleoproteins, and phospholipids. It remains controversial whether NAA contribute to or protect from autoimmune diseases. Using site-directed transgenic (sd-tg) mice expressing a prototypic NAA, we investigated the effect of NAA and NAA-producing B cells in disease development in the autoimmune-prone MRL/MpJ-Fas(lpr) (MRL-lpr) mice. We found that the expression of NAA in MRL-lpr mice prevented proteinuria and reduced kidney immune complex formation. The mice had significantly improved survival. Administration of the IgM NAA to MRL-lpr mice also delayed the onset of nephritis. The sd-tg MRL-lpr mice had decreased levels of anti-dsDNA Abs, anti-Hep2 nuclear Abs, and anti-Sm/ribonucleoprotein Abs. There is a shift in the IgG subclass profile from IgG2a and IgG3 to IgG1 in the sd-tg MRL-lpr mice. The CD4(+) T cells from the sd-tg MRL-lpr mice had increased expression of the negative costimulatory molecule CTLA-4 and increased production of IL-10 as compared with those from the wild-type mice. Furthermore, the NAA B cells produced large amounts of IL-10 upon TLR stimulation. These results indicate that NAA and NAA-producing B cells play an important role in protection from lupus nephritis and suggest that the NAA B cells may have an immune regulatory function via the provision of IL-10.     

Cell cycle-dependent expression of cyclooxygenase-2 in human fibroblasts.

The purpose of this investigation is to determine whether the levels of cyclooxygenase-2 (COX-2) expression are cell cycle dependent. We used a serum-starved human foreskin fibroblast model to determine changes in COX-2 mRNA, protein, and promoter activity in response to stimulation with interleukin-1b (IL-1b) and phorbol 12-myristate 13-acetate (PMA) at G0, G1, S and G2/M phases of the cell cycle. IL-1b (1 ng/ml) and PMA (100 nM) induced robust COX-2 expression in the G0 cells, and the level of COX-2 expression declined progressively after the cells had entered the cell cycle. The COX-2 mRNA level at G1, S and G2/M phases of the cell cycle was 76%, 46%, and 30% of that at G0, respectively. A 5-flanking promoter fragment of COX-2 constructed into a luciferase expression vector was transfected into cells. The promoter activity in response to PMA stimulation was significantly higher in G0 than in S phase cells. These results imply that G0 cells are the key players in inflammation and other COX-2-dependent pathophysiological processes. When the cells are in the proliferative phase, COX-2 inducibility becomes restrained probably by an endogenous control mechanism to avoid COX-2 mediated oxidative DNA damage.     

COX-2 regulates the proliferation of glioma stem like cells

Cancer stem-like cells (CSCs) possessing features of neural precursor cells (NPC) influence initiation, recurrence and chemoresistance of glioblastoma multiforme (GBM). As inflammation is crucial for glioblastoma progression we investigated the effect of chronic IL-1β treatment on CSCs derived from glioblastoma cell line U87MG. Exposure to IL-1β for 10 days increased (i) accumulation of 8-OHdG - a key biomarker of oxidative DNA damage; (ii) DNA damage response (DDR) indicators γH2AX, ATM and DNA-PK; (iii) nuclear and cytoplasmic p53 and COX-2 levels and (iv) interaction between COX-2 and p53. Despite upregulating p53 expression IL-1β had no effect on cell cycle progression, apoptosis or self renewal capacity of CSCs. COX-2 inhibitor Celecoxib reduced self renewal capacity and increased apoptosis of both control and IL-1β treated CSCs. Therefore the ability of COX-2 to regulate proliferation of CSCs irrespective of exposure to IL-1β, warrants further investigation of COX-2 as a potential anti-glioma target.     

Interleukin-4 induces specific pp-GalNAc-T expression and alterations in mucin O-glycosylation in colonic epithelial cells

Mucus hypersecretion occurs as a consequence of the Th2 immune response in epithelia, yet it was not previously known whether the degree of O-glycosylation was modulated under such conditions. A colonic carcinoma cell line LS174T was used to assess the effect of interleukin (IL)-4 on the mRNA levels of eight pp-GalNAc-Ts. A three- to four-fold increase in pp-GalNAc-T1, T4, and T7 levels was observed. Lysates of untreated or IL-4-treated cells were examined for their ability to transfer GalNAc residues onto a peptide corresponding to the tandem repeat portion of human MUC2. The number of incorporated GalNAc residues was greater after incubation with lysates of IL-4-treated cells than with lysates of untreated cells. Mucin-like large glycoproteins secreted by IL-4-treated cells had higher binding capacity to PNA and VVA-B₄ than those secreted by untreated cells. The results indicated that IL-4-treated LS174T cells are able to produce mucins with a higher degree of O-glycosylation than untreated counterparts.