Cytokine-induced gene expression of a neutrophil chemotactic factor/IL-8 in human hepatocytes

The liver participates in inflammation via the elaboration of acute phase proteins from hepatocytes in response to IL-1, TNF-alpha, and IL-6/INF-beta 2/hepatocyte-stimulating factor. In addition, some inflammatory states of the liver are characterized by leukocyte infiltrates. Here we demonstrate that human hepatocyte lines are capable of expressing mRNA and biologic activity for a neutrophil chemotactic factor (NCF)/IL-8 in response to the inflammatory mediators IL-1 alpha, IL-1 beta, and TNF. Two human hepatoma cell lines (SK-Hep and Hep-G2) displayed a time- and dose-dependent increase in steady state levels of NCF/IL-8 mRNA and secretion of chemotactic activity in response to TNF and IL-1. Neutralizing antibody to NCF/IL-8 inhibited hepatocyte-derived chemotactic activity by 88%. In contrast to IL-1 and TNF, hepatocytes did not respond to LPS or IL-6 within the time and dose parameters used above. Although the expression of NCF/IL-8 mRNA (1.8 kb) was first detectable between 1 and 2 h poststimulation, significant chemotactic bioactivity was not observed until about 4 h. Heat-inactivated (100 degrees C, 30 min) cytokine failed to induced NCF/IL-8 mRNA synthesis, and cotreatment of cells with cytokine and cycloheximide super-induced NCF/IL-8 mRNA while inhibiting production of bioactivity. Thus, NCF/IL-8 expression is a primary induction phenomenon. Our data demonstrate the stimulus specific induction of NCF/IL-8 in hepatocytes and suggest that cytokine cell-to-cell communication circuits may be important in neutrophil-mediated inflammatory processes in the liver.     

Ethylene promotes submergence-induced expression of OsABA8ox1, a gene that encodes ABA 8'-hydroxylase in rice

A rapid decrease of the plant hormone ABA under submergence is thought to be a prerequisite for the enhanced elongation of submerged shoots of rice (Oryza sativa L.). Here, we report that the level of phaseic acid (PA), an oxidized form of ABA, increased with decreasing ABA level during submergence. The oxidation of ABA to PA is catalyzed by ABA 8'-hydroxylase, which is possibly encoded by three genes (OsABA8ox1, -2 and -3) in rice. The ABA 8'-hydroxylase activity was confirmed in microsomes from yeast expressing OsABA8ox1. OsABA8ox1-green fluorescent protein (GFP) fusion protein in onion cells was localized to the endoplasmic reticulum. The mRNA level of OsABA8ox1, but not the mRNA levels of other OsABA8ox genes, increased dramatically within 1 h after submergence. On the other hand, the mRNA levels of genes involved in ABA biosynthesis (OsZEP and OsNCEDs) decreased after 1-2 h of submergence. Treatment of aerobic seedlings with ethylene and its precursor, 1-aminocyclopropane-1-carboxylate (ACC), rapidly induced the expression of OsABA8ox1, but the ethylene treatment did not strongly affect the expression of ABA biosynthetic genes. Moreover, pre-treatment with 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene action, partially suppressed induction of OsABA8ox1 expression under submergence. The ABA level was found to be negatively correlated with OsABA8ox1 expression under ACC or 1-MCP treatment. Together, these results indicate that the rapid decrease in ABA levels in submerged rice shoots is controlled partly by ethylene-induced expression of OsABA8ox1 and partly by ethylene-independent suppression of genes involved in the biosynthesis of ABA.     

Trypanosomal immunosuppressive factor: a secretion product(s) of Trypanosoma cruzi that inhibits proliferation and IL-2 receptor expression by activated human peripheral blood mononuclear cells

Coculture of blood forms of Trypanosoma cruzi with human PMBC suppresses the expression of several molecules involved in lymphocyte activation, including receptors for IL-2. Our work was initially undertaken to establish whether this effect required physical parasite-PBMC contact or was mediated by a T. cruzi secretion product. Using culture inserts with cell-impermeable membranes, we were able to demonstrate significant suppression of PHA-induced lymphoproliferation whether the trypanosomes were placed in the same compartment as, or separated from, the PBMC. Similar effects were observed by using supernatants from T. cruzi suspensions. These supernatants, which we refer to as trypanosomal immunosuppressive factor, also inhibited IL-2R expression in response to PHA stimulation. The suppressive effect of the secretion product(s) of T. cruzi was reversible, as evidenced by significant recovery of the proliferative capacity of PBMC after removal of the parasite-containing inserts. Moreover, the extent of the suppression produced by trypanosomal immunosuppressive factor subsided as culture time increased. Treatment of trypanosomal immunosuppressive factor with proteases abrogated its suppressive activity, suggesting that the relevant principle(s) was of protein nature. From ultrafiltration experiments, the molecular mass of the suppressive molecule(s) was estimated to be between 30,000 and 100,000 Da. These results demonstrate for the first time the capacity of T. cruzi to spontaneously secrete a factor that suppresses human lymphocyte responses in vitro. This factor, which may play a role in the down-regulation of host immune function observed in acute chagasic patients, might be a useful tool in exploring the mechanisms that regulate the expression of IL-2R and other surface molecules playing key roles in lymphocyte activation.     

Butyrate inhibits IL-1β-induced inflammatory gene expression by suppression of NF-κB activity in pancreatic beta cells

Cytokine-induced beta cell dysfunction is a hallmark of type 2 diabetes (T2D). Chronic exposure of beta cells to inflammatory cytokines affects gene expression and impairs insulin secretion. Thus, identification of anti-inflammatory factors that preserve beta cell function represents an opportunity to prevent or treat T2D. Butyrate is a gut microbial metabolite with anti-inflammatory properties for which we recently showed a role in preventing interleukin-1β (IL-1β)-induced beta cell dysfunction, but how prevention is accomplished is unclear. Here, we investigated the mechanisms by which butyrate exerts anti-inflammatory activity in beta cells. We exposed mouse islets and INS-1E cells to a low dose of IL-1β and/or butyrate and measured expression of inflammatory genes and nitric oxide (NO) production. Additionally, we explored the molecular mechanisms underlying butyrate activity by dissecting the activation of the nuclear factor-κB (NF-κB) pathway. We found that butyrate suppressed IL-1β-induced expression of inflammatory genes, such as Nos2, Cxcl1, and Ptgs2, and reduced NO production. Butyrate did not inhibit IκBα degradation nor NF-κB p65 nuclear translocation. Furthermore, butyrate did not affect binding of NF-κB p65 to target sequences in synthetic DNA but inhibited NF-κB p65 binding and RNA polymerase II recruitment to inflammatory gene promoters in the context of native DNA. We found this was concurrent with increased acetylation of NF-κB p65 and histone H4, suggesting butyrate affects NF-κB activity via inhibition of histone deacetylases. Together, our results show butyrate inhibits IL-1β-induced inflammatory gene expression and NO production through suppression of NF-κB activation and thereby possibly preserves beta cell function.     

Partial silencing of fucosyltransferase 8 gene expression inhibits proliferation of Ishikawa cells,a cell line of endometrial cancer

Endometrial cancer is the most common gynecologic malignancy and is associated with increased morbidity each year, including young people. However, its mechanisms of proliferation and progression are not fully elucidated. It is well known that abnormal glycosylation is involved in oncogenesis, and fucosylation is one of the most important types of glycosylation. In particular, fucosyltransferase 8 (FUT8) is the only FUT responsible for α1, 6-linked fucosylation (core fucosylation), and it is involved in various physiological as well as pathophysiological processes, including cancer biology. Therefore, we aimed to identify the expression of FUT8 in endometrial endometrioid carcinoma and investigate the effect of the partial silencing of the FUT8 gene on the cell proliferation of Ishikawa cells, an epithelial-like endometrial cancer cell line. Quantitative real-time PCR analysis showed that FUT8 gene expression was significantly elevated in the endometrial endometrioid carcinoma, compared to the normal endometrium. The immunostaining of FUT8 and Ulex europaeus Agglutinin 1 (UEA-1), a kind of lectin family specifically binding to fucose, was detected endometrial endometrioid carcinoma. The proliferation assay showed FUT8 partial knockdown by transfection of siRNA significantly suppressed the proliferation of Ishikawa cells, concomitant with the upregulation in the gene expressions associated with the interesting pathways associated with de-ubiquitination, aspirin trigger, mesenchymal-epithelial transition (MET) et al. It was suggested that the core fucosylation brought about by FUT8 might be involved in the proliferation of endometrial endometrioid carcinoma cells.     

IL-8 and global gene expression analysis define a key role of ATP in renal epithelial cell responses induced by uropathogenic bacteria

The recent recognition of receptor-mediated ATP signalling as a pathway of epithelial pro-inflammatory cytokine release challenges the ubiquitous role of the TLR4 pathway during urinary tract infection. The aim of this study was to compare cellular responses of renal epithelial cells infected with uropathogenic Escherichia coli (UPEC) strain IA2 to stimulation with ATP-γ-S. A498 cells were infected or stimulated in the presence or absence of apyrase, that degrades extracellular ATP, or after siRNA-mediated knockdown of ATP-responding P2Y₂ receptors. Cellular IL-8 release and global gene expression were analysed. Both IA2 and A498 cells per se released ATP, which increased during infection. IA2 and ATP-γ-S caused a ∼5-fold increase in cellular release of IL-8 and stimulations performed in the presence of apyrase or after siRNA knockdown of P2Y₂ receptors resulted in attenuation of IA2-mediated IL-8 release. Microarray results show that both IA2 and ATP-γ-S induced marked changes in gene expression of renal cells. Thirty-six genes were in common between both stimuli, and many of these are key genes belonging to classical response pathways of bacterial infection. Functional analysis shows that 88 biological function-annotated cellular pathways were identical between IA2 and ATP-γ-S stimuli. Results show that UPEC-induced release of IL-8 is dependent on P2Y₂ signalling and that cellular responses elicited by UPEC and ATP-γ-S have many identical features. This indicates that renal epithelial responses elicited by bacteria could be mediated by bacteria- or host-derived ATP, thus defining a key role of ATP during infection.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9414-7) contains supplementary material, which is available to authorized users.