Effect of IL-1alpha on prostaglandin synthesis of oestrogenized rat uterus is mediated by nitric oxide

We examined the possible relationship between cytokines, nitric oxide (NO) and prostaglandins in the oestrogenized rat uterus. Results indicate that: IL-1alpha but not IL-2 enhances the synthesis of prostaglandins in oestrogenized rat uteri; IL-1alpha but not IL-2 induced an augmention of NO production in this tissue; the effect of IL-1alpha on prostaglandin synthesis is abolished by NMMA, an NO antagonist; NS-398, a COX-2 inhibitor, prevents the augmention of prostaglandins produced by IL-1alpha. These results suggest that there is an interaction between IL-1alpha, NO and prostaglandins and that this interrelationship involves COX-2. This mechanism might be important during implantation and labor.     

c-Jun contributes to amyloid beta-induced neuronal apoptosis but is not necessary for amyloid beta-induced c-jun induction

The role of gene expression in neuronal apoptosis may be cell- and apoptotic stimulus-specific. Previously, we and others showed that amyloid beta (Abeta)-induced neuronal apoptosis is accompanied by c-jun induction. Moreover, c-Jun contributes to neuronal death in several apoptosis paradigms involving survival factor withdrawal. To evaluate the role of c-Jun in Abeta toxicity, we compared Abeta-induced apoptosis in neurons from murine fetal littermates that were deficient or wild-type with respect to c-Jun. We report that neurons deficient for c-jun are relatively resistant to Abeta toxicity, suggesting that c-Jun contributes to apoptosis in this model. When changes in gene expression were quantified in neurons treated in parallel, we found that Abeta treatment surprisingly led to an apparent activation of the c-jun promoter in both the c-jun-deficient and wild-type neurons, suggesting that c-Jun is not necessary for activation of the c-jun promoter. Indeed, several genes induced by Abeta in wild-type neurons were also induced in c-jun-deficient neurons, including c-fos, fosB, ngfi-B, and ikappaB. In summary, these results indicate that c-Jun contributes to Abeta-induced neuronal death but that c-Jun is not necessary for c-jun induction.     

Fructan synthesis is inhibited by phosphate in warm-grown, but not in cold-treated, excised barley leaves

The inhibition of fructan accumulation by phosphate was investigated in warm-grown and cold-treated barley (Hordeum vulgare) plants. Detached leaves were incubated in water or phosphate for 24 h under lighting or in darkness. Fructosyltransferase, sucrose phosphate synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (FBPase) activities were subsequently analysed, as well as the content of carbohydrates, hexose-phosphates, phosphate, amino acids and protein. In warm-grown leaves, phosphate decreased fructan accumulation and total carbon in carbohydrates and did not affect protein content. Phosphate increased hexose-phosphates, phosphate and amino acids. Fructosyltransferase and FBPase activities were not affected by phosphate feeding, while SPS activity was inhibited by phosphate in incubations in both light and darkness. In cold-treated leaves, which before incubation had higher SPS activities than warm-grown leaves, phosphate had no inhibitory effect on fructan accumulation, carbohydrate content or total C in carbohydrates. The activities of SPS and FBPase were unaffected by phosphate. The results indicate that phosphate decreases fructan accumulation through an inhibition of SPS whenever this activity is not high before a rise in phosphate content.     

A phosphatidylserine species inhibits a range of TLR- but not IL-1beta-induced inflammatory responses by disruption of membrane microdomains

TLRs detect conserved molecular patterns that are unique to microbes, enabling tailored responses to invading pathogens and modulating a multitude of immunopathological conditions. We investigated the ability of a naturally occurring stearoyl-arachidonoyl form of phosphatidylserine (SAPS) to inhibit the proinflammatory effects of TLR agonists in models of inflammation investigating the interaction of leukocytes with epithelial and endothelial cells. The responses to LPS of both epithelial and endothelial cells were highly amplified in the presence of PBMCs. Coincubation with SAPS markedly inhibited activation of cocultures by LPS, principally through inhibition of the TLR4 signaling pathway in PBMCs; however, this was not through downmodulation of TLR4 or coreceptor expression, nor was IL-1beta-induced cytokine release affected. SAPS also impaired Pam(3)CSK(4) (TLR2/1), Gardiquimod (TLR7/8), and Streptococcus pneumoniae-induced cytokine release, but had only modest effects on poly(I:C) (TLR3)-induced responses. Fluorescence resonance energy transfer analysis of molecular associations revealed that SAPS disrupted the association of both TLR4 and TLR2 with their respective membrane partners that are required for signaling. Thus, our data reinforce the existence and importance of cooperative networks of TLRs, tissue cells, and leukocytes in mediating innate immunity, and identify a novel disrupter of membrane microdomains, revealing the dependence of TLR signaling on localization within these domains.     

Monocyte suppressor factor is plasminogen activator inhibitor inhibition of membrane bound but not soluble IL-1

Human monocytes cultured in monolayer for 6 days were found to secrete a factor that suppressed the T cell proliferative response to soluble Ag and to alloantigens. The elaboration of this monocyte suppressor factor (MSF) was not inhibited by indomethacin. It has an apparent Mr of 50 to 60 kDa. It does not inhibit soluble IL-1 in the murine thymocyte costimulator assay but does inhibit the activity of membrane bound IL-1, which we observed to be almost exclusively IL-1 alpha. MSF contains elevated amounts of plasminogen activator inhibitor (PAI) when measured either as bioactivity or in an ELISA. Its immunosuppressive properties are inhibited by anti-PAI antibody. Furthermore, the eluate but not the effluent of an anti-PAI immunoabsorbent column contains all of the immunosuppressive activity. Based on these data we suggest that MSF is, in fact, PAI and postulate that the mechanism of action is inhibition of the plasmin cascade, thereby preventing the release of membrane bound IL-1. This suggests that monocytes possess an autoregulatory circuit that may have implications for the kinetics of the inflammatory response.     

The induction of cyclooxygenase-2 in IL-1beta-treated endothelial cells is inhibited by prostaglandin E2 through cAMP

Prostaglandins (PGs) have numerous cardiovascular and inflammatory effects. Cyclooxygenase (COX), which exists as COX-1 and COX-2 isoforms, is the first enzyme in the pathway in which arachidonic acid is converted to PGs. Prostaglandin E2 (PGE2) exerts a variety of biological activities for the maintenance of local homeostasis in the body. Elucidation of PGE2 involvement in the signalling molecules such as COX could lead to potential therapeutic interventions. Here, we have investigated the effects of PGE2 on the induction of COX-2 in human umbilical vein endothelial cells (HUVEC) treated with interleukin-1beta (IL-1beta 1 ng/ml). COX activity was measured by the production of 6-keto-PGF1alpha, PGE2, PGF2alpha and thromboxane B2 (TXB2) in the presence of exogenous arachidonic acids (10 microM for 10 min) using enzyme immunoassay (EIA). COX-1 and COX-2 protein was measured by immunoblotting using specific antibody. Untreated HUVEC contained only COX-1 protein while IL-1beta treated HUVEC contained COX-1 and COX-2 protein. PGE2 (3 microM for 24h) did not affect on COX activity and protein in untreated HUVEC. Interestingly, PGE2 (3 microM for 24h) can inhibit COX-2 protein, but not COX-1 protein, expressed in HUVEC treated with IL-1beta. This inhibition was reversed by coincubation with forskolin (100 microM). The increased COX activity in HUVEC treated with IL-1beta was also inhibited by PGE2 (0.03, 0.3 and 3 microM for 24h) in a dose-dependent manner. Similarly, forskolin (10, 50 or 100 microM) can also reverse the inhibition of PGE2 on increased COX activity in IL-1beta treated HUVEC. The results suggested that (i) PGE2 can initiate negative feedback regulation in the induction of COX-2 elicited by IL-1beta in endothelial cells, (ii) the inhibition of PGE2 on COX-2 protein and activity in IL-1beta treated HUVEC is mediated by cAMP and (iii) the therapeutic use of PGE2 in the condition which COX-2 has been involved may have different roles.     

Dimerization of corticotropin-releasing factor receptor type 1 is not coupled to ligand binding

As described previously, receptor dimerization of G protein-coupled receptors may influence signaling, trafficking, and regulation in vivo. Up to now, most studies aiming at the possible role of receptor dimerization in receptor activation and signal transduction are focused on class A GPCRs. In the present work, the dimerization behavior of the corticotropin-releasing factor receptor type 1 (CRF1R), which belongs to class B of GPCRs and plays an important role in coordination of the immune response, stress, and learning behavior, was investigated by using fluorescence resonance energy transfer (FRET). For this purpose, we generated fusion proteins of CRF1R tagged at their C-terminus to a cyan or yellow fluorescent protein, which can be used as a FRET pair. Binding studies verified that the receptor constructs were able to bind their natural ligands in a manner comparable with the wild-type receptor, whereas cAMP accumulation proved the functionality of the constructs. In microscopic studies, a dimerization of the CRF1R was observed, but the addition of either CRF-related agonists or antagonists did not show any dose-related increase of the observed FRET signal, indicating that the dimer-monomer ratio is not changed on addition of ligand.     

Endoreduplication is not inhibited but induced by aphidicolin in cultured cells of tobacco

Endoreduplication is a common process in plants that allows cells to increase their DNA content. In the tobacco cell cultures studied in this work it can be induced by simple hormone deprivation. Mesophyll protoplast-derived cells cultured in the presence of NAA (auxin) and BAP (cytokinin) keep on dividing, while elongation and concomitant DNA endoreduplication are induced and maintained in a medium containing only NAA. If aphidicolin is given to the two types of culture, no effect is observed on elongating, endoreduplicating cells. However, the cells programmed for division switch to elongation and DNA endoreduplication. Thus aphidicolin, an inhibitor of the replicative DNA polymerases, alpha and delta, does not inhibit endoreduplication, and furthermore actually induces it when the mitotic cell cycle is blocked. DNA duplication and cell growth can only be completely blocked if ddTTP, an inhibitor of DNA polymerase-beta, is given together with aphidicolin. This result implies that an aphidicolin-resistant DNA polymerase, such as the repair-associated DNA polymerase-beta, can mediate DNA synthesis during endoreduplication and can substitute for polymerases-alpha and -delta when the latter are inhibited. Similar results are obtained in cultures of the BY-2 cell line by withdrawing auxins from the culture medium. In this cell line endoreduplication is induced only in a small proportion of the cells. A greater proportion of the cells are blocked in the G(2) phase of the cell cycle.     

Calcium diphosphatidate membrane traversal is inhibited by common phospholipids and cholesterol but not by plasmalogen

Phosphatidate-mediated Ca2+ membrane traversal is inhibited by phospholipids (PL) such a phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin and lysoPC, but not by PC-plasmalogen. Kinetics of Ca2+ traversal through a 'passive' bilayer consisting of OH-blocked cholesterol show competition between PC and phosphatidic acid (PA); it appears likely that a Ca(PA.PC) complex is formed which is not a transmembrane ionophore but will reduce the amount of phosphatidic acid available for the formation of the ionophore, Ca(PA)2. PS and PI may inhibit Ca2+-traversal in the same manner by forming Ca(PA.PL) complexes. We suggest that PC-plasmalogen, with one of the Ca2+-chelating ester CO groups missing, cannot engage in calcium cages, i.e., Ca(PA.PL) complexes, and thus does not interfere with Ca(PA)2 formation. Double-reciprocal plotting of Ca2+ traversal rates in cholesterol-containing liposomes vs. calcium concentration suggests that cholesterol inhibits Ca2+ traversal by competing with Ca2+ for PA. The inhibition does not seem to be caused by a restructuring or dehydration of the membrane 'hydrogen belts' affected by cholesterol; most probably, it is due to hydrogen bonding of the cholesterol-OH group to a CO group of PA; this reduces the amount of PA available for the calcium ferry. The inhibition by sphingomyelin and lysoPC may also be explained by their OH group interacting with PA via hydrogen bonding. The pH dependence of Ca2+ traversal suggests that H[Ca(PA)2]- can serve as Ca2+ cross-membrane ferry but that at physiological pH, [Ca(PA)2]2- is the predominant ionophore. In conclusion, the results indicate that Ca2+ traversal is strongly dependent on the structure of the hydrogen belts, i.e., the membrane strata occupied by hydrogen bond acceptors (CO of phospholipids) and donors (OH of cholesterol, sphingosine), and that lipid hydrogen belt structures may regulate storage and passage of Ca2+.     

TAB1 modulates IL-1alpha mediated cytokine secretion but is dispensable for TAK1 activation

Biochemical evidence indicates that TGF-beta-activated kinase 1 (TAK1), a key modulator of the inflammatory response, exists in a complex with various adaptor proteins including the TAK1 binding protein 1 (TAB1). However, the physiological importance of TAB1 in TAK1 activation, and in the subsequent induction of proinflammatory mediators, remains unclear. In this study, a critical role for TAK1 in IL-1alpha or TNFalpha stimulated MAPK and NFkappaB activation was confirmed by inhibition of the nuclear accumulation of NFkappaB p65 and phosphorylated forms of c-Jun and p38 following siRNA mediated TAK1 silencing. These effects were associated with significant reductions in IL-1alpha stimulated levels of secreted IL-6, IL-8, MCP-1 and GM-CSF. In contrast, IL-1alpha or TNFalpha dependent cellular redistribution of NFkappaB p65 and phosphorylated c-Jun and p38 was not affected by 80% siRNA mediated knockdown of TAB1 protein levels. Interestingly, IL-6, IL-8 and GM-CSF release from TAB1 siRNA transfected cells was significantly reduced following IL-1alpha treatment, but was unchanged after TNFalpha stimulation, suggesting differential roles for TAB1 in IL-1alpha and TNFalpha signalling pathways. These findings may imply an as yet unidentified role for TAB1 in the inflammatory response, which is independent of the activation of classical TAK1 associated signalling cascades.     

Tumor necrosis factor alpha-induced inflammation is increased but apoptosis is inhibited by common food additive carrageenan

Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, is secreted in response to inflammatory stimuli in diseases such as rheumatoid arthritis and inflammatory bowel disease. TNF-α mediates both apoptosis and inflammation, stimulating an inflammatory cascade through the non-canonical pathway of NF-κB activation, leading to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN) stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activation and utilizes the adaptor molecule BCL10 (B-cell leukemia/lymphoma 10). In a series of experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNF-α and carrageenan in order to simulate the possible effects of exposure to dietary CGN in the setting of a TNF-α-mediated inflammatory disease process. A marked increase in secretion of IL-8 occurred, attributable to synergistic effects on phosphorylated NF-κB-inducing kinase (NIK) in the non-canonical pathway. TNF-α induced the ubiquitination of TRAF2 (TNF receptor-associated factor 2), which interacts with NIK, and CGN induced phosphorylation of BCL10, leading to increased NIK phosphorylation. These results suggest that TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNF-α, including activation of caspase-8 and PARP-1 (poly(ADP-ribose) polymerase 1) fragmentation, were markedly reduced in the presence of CGN, and CGN caused reduced expression of Fas. These findings demonstrate that exposure to CGN drives TNF-α-stimulated cells toward inflammation rather than toward apoptotic cell death and suggest that CGN exposure may compromise the effectiveness of anti-TNF-α therapy.     

Necrosis is associated with IL-6 production but apoptosis is not

Due to loss of cell membrane integrity, necrotic cells passively release several cytosolic factors that can activate antigen presenting cells and other immune cells. In contrast, cells dying by apoptosis do not induce an inflammatory response. Here we show that necrotic cell death induced by several stimuli, such as TNF, anti-Fas or dsRNA, coincides with NF-kappaB-and p38MAPK-mediated upregulation and secretion of the pro-inflammatory cytokine IL-6. This event is greatly reduced or absent in conditions of apoptotic cell death induced by the same stimuli. This demonstrates that besides the capacity of necrotic cells to induce an inflammatory response due to leakage of cellular contents, necrotic dying cells themselves are involved in the expression and secretion of inflammatory cytokines. Moreover, inhibition of NF-kappaB and p38MAPK activation does not affect necrotic cell death in all conditions tested. This suggests that the activation of inflammatory pathways is distinct from the activation of necrotic cell death sensu strictu.     

Interleukin-1 beta-induced anorexia is reversed by ghrelin

Interleukins, in particular interleukin-1β (IL-1β), reduce food intake after peripheral and central administration, which suggests that they contribute to anorexia during various infectious, neoplastic, and autoimmune diseases. On the other hand, ghrelin stimulates food intake by acting on the central nervous system (CNS) and is considered an important regulator of food intake in both rodents and humans. In the present study, we investigated if ghrelin could reverse IL-1β-induced anorexia. Intracerebroventricular (i.c.v.) injection of 15, 30 or 45 ng/μl of IL-1β caused significant suppression of food intake in 20 h fasting animals. This effect lasted for a 24 h period. Ghrelin (0.15 nmol or 1.5 nmol/μl) produced a significant increase in cumulative food intake in normally fed animals. However, it did not alter food intake in 20 h fasting animals. Central administration of ghrelin reduced the anorexic effect of IL-1β (15 ng/μl). The effect was observed 30 min after injection and lasted for the next 24 h. This study provides evidence that ghrelin is an orexigenic peptide capable of antagonizing IL-1β-induced anorexia.     

Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, but not by TNF-alpha

Synthesis of the antimicrobial protein neutrophil gelatinase-associated lipocalin (NGAL) increases dramatically in bronchial epithelial cells and alveolear type II pneumocytes during lung inflammation. IL-1beta induces a >10-fold up-regulation of NGAL expression in the type II pneumocyte-derived cell line A549 cells, whereas TNF-alpha, IL-6, and LPS had no effect. Similar IL-1beta selectivity was demonstrated in primary bronchial epithelial cells and epidermal keratinocytes and for an NGAL promoter fragment transfected into A549 cells. By deletion and substitution analysis of the NGAL promoter, a 40-bp region containing an NF-kappaB consensus site was found to control the IL-1beta-specific up-regulation. Involvement of the NF-kappaB site was demonstrated by site-directed mutagenesis, by transfection with a dominant-negative inhibitor of the NF-kappaB pathway, and by EMSA. TNF-alpha activation of NF-kappaB, in contrast, did not increase NGAL synthesis, even though induced binding of NF-kappaB to the NGAL promoter was observed in vitro. IL-1beta specificity was not contained within the NF-kappaB site of the NGAL promoter, as determined by exchanging the NGAL promoter's NF-kappaB-binding sequence with that of the IL-8 promoter or with the NF-kappaB consensus sequence and by testing the NF-kappaB-binding sequence of the NGAL promoter against the heterologous SV40 promoter. Selectivity for the IL-1 pathway was substantiated by demonstrating that NGAL promoter activity could be induced by LPS stimulation of A549 cells transiently expressing Toll-like receptor 4, which use the same intracellular signaling pathway as the IL-1R. Together, this demonstrates a selective up-regulation of NGAL by the IL-1 pathway.     

Matriptase is inhibited by extravascular antithrombin in epithelial cells but not in most carcinoma cells

Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments where its target proteases are largely unknown. This serpin was previously detected in human milk as complexes with matriptase, a membrane-bound serine protease broadly expressed in epithelial and carcinoma cells, and under tight regulation by hepatocyte growth factor activator inhibitor (HAI)-1, a transmembrane Kunitz-type serine protease inhibitor that forms heat-sensitive complexes with active matriptase. In the current study, we detect, in addition to matriptase-HAI-1 complexes, heat-resistant matriptase complexes generated by nontransformed mammary, prostate, and epidermal epithelial cells that we show to be matriptase-antithrombin complexes. These findings suggest that in addition to HAI-1, interstitial antithrombin participates in the regulation of matriptase activity in epithelial cells. This physiological mechanism appears, however, to largely be lost in cancer cells since matriptase-antithrombin complexes were not detected in all but two of a panel of seven breast, prostate, and ovarian cancer cell lines. Using purified active matriptase, we further characterize the formation of matriptase-antithrombin complex and show that heparin can significantly potentiate the inhibitory potency of antithrombin against matriptase. Second-order rate constants for the inhibition were determined to be 3.9 × 10(3) M(-1)s(-1) in the absence of heparin and 1.2 × 10(5) M(-1)s(-1) in the presence of heparin, a 30-fold increase, consistent with the established role of heparin in activating antithrombin function. Taken together these data suggest that normal epithelial cells employ a dual mechanism involving HAI-1 and antithrombin to control matriptase and that the antithrombin-based mechanism appears lost in the majority of carcinoma cells.