Dietary nucleotides increase the mucosal IgA response and the secretion of transforming growth factor β from intestinal epithelial cells in mice

We have investigated the influence of dietary nucleotides on the intestinal immune system in ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic mice (OVA-TCR Tg mice). When mice were supplied with water supplemented with 2% OVA ad libitum, the faecal OVA-specific immunoglobulin A (IgA) level significantly increased in those fed a nucleotide-supplemented diet (NT(+) diet) compared with those fed a nucleotide-free control diet (NT(–) diet). In the NT(+) diet-fed mice, secretion of transforming growth factor β (TGF-β), which is an isotype-specific switch factor for IgA, from intestinal epithelial cells (IECs) was significantly increased. Furthermore, an increased proportion of intestinal intraepithelial lymphocytes (IELs) bearing γδ TCR (TCRγδ⁺ IELs) and increased secretion from IECs of interleukin 7 (IL-7), which is essential for the development of TCRγδ⁺ IELs, were also observed in OVA-TCR-Tg mice fed the NT(+) diet, as we previously demonstrated using BALB/c mice (Nagafuchi et al., Biosci. Biotechnol. Biochem. 64: 1459-65 (2000)). Considering that TCRγδ⁺ T cells and TGF-β are important for an induction of the mucosal IgA response, our results suggest that dietary nucleotides augment the mucosal OVA-specific IgA response by increasing the secretion of TGF-β from IECs and the proportion of TCRγδ⁺ IELs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transforming growth factor-β1 enhances the secretion of monocyte chemoattractant protein-1 by the IEC-18 intestinal epithelial cell line

Summary Intestinal epithelial cells (IEC) are known to produce monocyte chemoattractant protein-1 (MCP-1). However, MCP-1 production, as with many other cytokines, can be regulated by a network of cytokines present in the environment of the IEC. Both IEC and inflammatory cells have been shown to produce transforming growth factor-β (TGF-β), and the regulatory effect of this cytokine on MCP-1 secretion by IEC has not been determined. Using the IEC-18 cell line, we have found that TGF-β1 alone induced the secretion of high levels of MCP-1. Treatment with TGF-β1 also enhanced the levels of MCP-1 messenger ribonucleic acid. However, costimulation of the cells with TGF-β1 and interleukin-1β (IL-1β) resulted in significant, but less than additive, increases in MCP-1 secretion. Finally, the enhancing effect of TGF-β1 on MCP-1 secretion was not due to IL-6. These results suggest that TGF-β1 from IEC or inflammatory cells may significantly enhance the secretion of MCP-1 by IEC and play an important role in inflamed mucosal tissues.     

Transforming growth factor-β1 (TGF-β1) and acetylcholine (ACh) alter nitric oxide (NO) and interleukin-1β (IL-1β) secretion in human colon adenocarcinoma cells

Colon adenocarcinoma is one of the most common fatal malignancies in Western countries. Progression of this cancer is dependent on tumor microenvironmental signaling molecules such as transforming growth factor-β (TGF-β) or acetylcholine (ACh). The present study was conducted to assess the influence of recombinant human transforming growth factor (rhTGF)-β1 or ACh on nitric oxide (NO) and interleukin-1β (IL-1β) secretion by three human colon adenocarcinoma cell lines: HT29, LS180, and SW948, derived from different grade tumors (Duke’s stage). The cells were cultured in 2D and 3D (spheroids) conditions. Colon carcinoma cells exhibited different sensitivities to rhTGF-β1 or ACh dependent on the tumor grade and the culture model. ACh exhibited significant inhibitory effects towards NO, endothelial nitric oxide synthase (eNOS), and IL-1β secretion especially by tumor cells derived form Duke’s C stage of colon carcinoma. rhTGF-β1 also decreased NO, IL-1β, and eNOS expression, but its effect was lower than that observed after the administration of ACh. The inhibition of NO and IL-1β production was more striking in 3D tumor spheroids than in 2D culture monolayers. Taken together, the TGF-β1–ACh axis may regulate colon carcinoma progression and metastasis by altering NO secretion and influence inflammatory responses by modulating IL-1β production.     

The effects of TGF-β1 on the expression of type IV collagenases in mouse peritoneal macrophages

Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that plays a critical role in modulating immune response and inflammation. We have investigated the effects of TGF-β1 on the expression of type IV collagenases, matrix metalloproteinase (MMP)-2 and MMP-9, in mouse peritoneal macrophages. TGF-β1 alone enhanced the secretion of MMP-9, while it blocked lipopolysaccharide (LPS)-stimulated MMP-9 production. We have shown that this biphasic effect of TGF-β1 is exerted at the mRNA level of the MMP-9 gene. Although TGF-β1 increased both basal and LPS-induced MMP-2 production at the protein and mRNA levels, the extent of the increase was smaller in LPS-activated macrophages than in control macrophages. The expression of type I and type II receptors for TGF-β was significantly decreased upon activation, suggesting that the lesser effect of TGF-β1 in activated macrophages may result from the decreased expression of TGF-β receptors. In addition, the expression of endogenous TGF-β1 mRNA was decreased significantly in activated macrophages. These findings suggest that activated macrophages not only produce less TGF-β1, but also respond less well to TGF-β to provide for inflammatory response.     

Cytokine production by a megakaryocytic cell line

Summary The regulation of megakaryopoeisis by cytokines is not yet well understood. It is possible that autocrine loops are established during megakaryocyte growth and differentiation, aiding in the maturation of these cells. The CHRF-288-11 human megakaryoblastic cell line has been examined for cytokine production in growing cells and cells stimulated to differentiate by the addition of phorbol esters. It has been demonstrated that these cells produce RNA corresponding to the interleukins IL-1α, 1β, 3, 7, 8, and 11, granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), interferon-α (INF-α), and basic fibroblast growth factor (bFGF). Additionaly, RNA corresponding to the receptors for IL-6, GM-CSF, SCF, INF-α,β, bFGF, and monocyte colony stimulating factor (M-CSF) were also expressed by the cells. The receptor for TNF-α was detected immunologically. Analysis at the protein level demonstrated that significant amounts of INF-α, TNF-α, GM-CSF, SCF, IL-1α, and a soluble form of the IL-6 receptor were produced by the cells. Addition of phorbol esters to CHRF-288-11 cells enhances their megakaryocytic phenotype; such treatment also results in increased secretion of INF-α, TNF-α, and GM-CSF. These results suggest that potential autocrine loops are established during the differentiation of CHRF-288-11 cells, which may alter the capability of the cell to differentiate. These findings are similar to those recently obtained for marrow-derived megakaryocytes (Jiang et al.) suggesting that CHRF-288-11 cells provide a useful model system for the study of cytokine release during megakaryocyte differentiation.     

Th2 cytokines down-regulate TLR expression and function in human intestinal epithelial cells

TLRs serve important immune and nonimmune functions in human intestinal epithelial cells (IECs). Proinflammatory Th1 cytokines have been shown to promote TLR expression and function in IECs, but the effect of key Th2 cytokines (IL-4, IL-5, IL-13) on TLR signaling in IECs has not been elucidated so far. We stimulated human model IECs with Th2 cytokines and examined TLR mRNA and protein expression by Northern blotting, RT-PCR, real-time RT-PCR, Western blotting, and flow cytometry. TLR function was determined by I-kappaBalpha phosphorylation assays, ELISA for IL-8 secretion after stimulation with TLR ligands and flow cytometry for LPS uptake. IL-4 and IL-13 significantly decreased TLR3 and TLR4 mRNA and protein expression including the requisite TLR4 coreceptor MD-2. TLR4/MD-2-mediated LPS uptake and TLR ligand-induced I-kappaBalpha phosphorylation and IL-8 secretion were significantly diminished in Th2 cytokine-primed IECs. The down-regulatory effect of Th2 cytokines on TLR expression and function in IECs also counteracted enhanced TLR signaling induced by stimulation with the hallmark Th1 cytokine IFN-gamma. In summary, Th2 cytokines appear to dampen TLR expression and function in resting and Th1 cytokine-primed human IECs. Diminished TLR function in IECs under the influence of Th2 cytokines may protect the host from excessive TLR signaling, but likely also impairs the host intestinal innate immune defense and increases IEC susceptibility to chronic inflammation in response to the intestinal microenvironment. Taken together, our data underscore the important role of Th2 cytokines in balancing TLR signaling in human IECs.     

Ecto-5′-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5′-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5′-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5′-AMP. EPEC infection triggered a release of ecto-5′-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5′-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5′-nucleotidase was susceptible to inhibition by zinc acetate and by α,β-methylene-adenosine diphosphate (α,β-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5′-AMP. In addition, α,β-methylene-ADP and zinc blocked the ability of 5′-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5′-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5′-nucleotidase, such as α,β-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.     

Putative implication of 3′-terminal segment of 18S rRNA in translation initiation of uncapped mRNAs in plants

A putative implication 3′-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3′-terminal segment (nucleotides 1777–1811) of 18S rRNA including the last hairpin 45 was accessible for complementary interactions within 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA, when added to wheat germ cell-free protein synthesizing system, specifically inhibited translation of uncapped reporter mRNA encoding β-glucuronidase. In the 5′-untranslated region (UTR), the reporter mRNA contained a leader sequence of potato virus Y (PVY) genomic RNA with fragments complementary to the region 1777–1811. A sequence corresponding to nucleotides 291–316 of PVY, which was complementary to most of the 3′-terminal 18S rRNA segment 1777–1808, was shown to enhance translational efficiency of the reporter mRNAs when placed into 5′-UTR. The obtained results suggest that complementary interactions between 5′-UTR of mRNA and 3′-terminal segment of 18S rRNA can take place during cap-independent translation initiation.     

Cytokine expression in cord blood cells of children of healthy and allergic mothers

To determine some early signs connected with the increased risk of future allergy development, gene expression and production of selected cytokines were tested in children of allergic mothers and compared with newborns of healthy mothers. Expression of IL-1β, IL-2, IL-4, IL-8, IL-10, IL-13, IFN-γ, TNF-α, TGF-β and EGF was tested in cord blood cells using real-time PCR and production of these cytokines was evaluated in cord sera by ELISA. Gene expression of IL-2, IL-4, IL-8, IFN-γ, IL-1β, TNF-α and TGF-β was decreased and that of IL-10, IL-13 and EGF increased in children of allergic mothers in comparison with those of healthy mothers. Significant differences in sera of healthy and allergic groups were only in IL-10 and EGF. Different relationship among serum cytokine levels reflects the fact that the cytokines are not produced only by blood cells. Significantly decreased production of EGF in newborns of allergic mothers could negatively influence maturation of mucosal membranes of these children and support thus their easier allergization. Allergic phenotype pointing to the bias to T_H2 response and to possibly impaired intestine maturation was apparent already on the level of cord blood and could serve as a predictive sign of increased allergy risk.     

Transforming growth factor β 869C/T and interleukin 6 -174G/C polymorphisms relate to the severity and progression of bone-erosive damage detected by ultrasound in rheumatoid arthritis

Introduction  

Single nucleotide polymorphisms (SNPs) of transforming growth factor β (TGF-β) and IL-6 genes (respectively, 869C/T and -174G/C) have been associated with radiographic severity of bone-erosive damage in patients with rheumatoid arthritis (RA). Musculoskeletal ultrasound (US) is more sensitive than radiography in detecting bone erosion. We analyzed the association between TGF-β 869C/T and IL-6 -174G/C SNPs and bone-erosive damage, evaluated by US, in a cohort of patients with severely active RA.     

TGF-β in progression of liver disease

Transforming growth factor-β (TGF-β) is a central regulator in chronic liver disease contributing to all stages of disease progression from initial liver injury through inflammation and fibrosis to cirrhosis and hepatocellular carcinoma. Liver-damage-induced levels of active TGF-β enhance hepatocyte destruction and mediate hepatic stellate cell and fibroblast activation resulting in a wound-healing response, including myofibroblast generation and extracellular matrix deposition. Being recognised as a major profibrogenic cytokine, the targeting of the TGF-β signalling pathway has been explored with respect to the inhibition of liver disease progression. Whereas interference with TGF-β signalling in various short-term animal models has provided promising results, liver disease progression in humans is a process of decades with different phases in which TGF-β or its targeting might have both beneficial and adverse outcomes. Based on recent literature, we summarise the cell-type-directed double-edged role of TGF-β in various liver disease stages. We emphasise that, in order to achieve therapeutic effects, we need to target TGF-β signalling in the right cell type at the right time.     

Lysozyme transgenic goats’ milk positively impacts intestinal cytokine expression and morphology

In addition to its well-recognized antimicrobial properties, lysozyme can also modulate the inflammatory response. This ability may be particularly important in the gastrointestinal tract where inappropriate inflammatory reactions can damage the intestinal epithelium, leading to significant health problems. The consumption of milk from transgenic goats producing human lysozyme (hLZ) in their milk therefore has the potential to positively impact intestinal health. In order to investigate the effect of hLZ-containing milk on the inflammatory response, young pigs were fed pasteurized milk from hLZ or non-transgenic control goats and quantitative real-time PCR was performed to assess local expression of TNF-α, IL-8, and TGF-β1 in the small intestine. Histological changes were also investigated, specifically looking at villi width, length, crypt depth, and lamina propria thickness along with cell counts for intraepithelial lymphocytes and goblet cells. Significantly higher expression of anti-inflammatory cytokine TGF-β1 was seen in the ileum of pigs fed pasteurized milk containing hLZ (P = 0.0478), along with an increase in intraepithelial lymphocytes (P = 0.0255), and decrease in lamina propria thickness in the duodenum (P = 0.0001). Based on these results we conclude that consuming pasteurized milk containing hLZ does not induce an inflammatory response and improves the health of the small intestine in pigs.     

Modulation of TGF-β-inducible hypermotility by EGF and other factors in human prostate epithelial cells and keratinocytes

Keratinocytes migrating from a wound edge or initiating malignant invasion greatly increase their expression of the basement membrane protein Laminin-322 (Lam332). In culture, keratinocytes initiate sustained directional hypermotility when plated onto an incompletely processed form of Lam332 (Lam332′) or when treated with transforming growth factor beta (TGF-β), an inducer of Lam332 expression. The development and tissue architecture of stratified squamous and prostate epithelia are very different, yet the basal cells of both express p63, α6β4 integrin, and Lam332. Keratinocytes and prostate epithelial cells grow well in nutritionally optimized culture media with pituitary extract and certain mitogens. We report that prostate epithelial cells display hypermotility responses indistinguishable from those of keratinocytes. Several culture medium variables attenuated TGF-β-induced hypermotility, including Ca⁺⁺, serum, and some pituitary extract preparations, without impairing growth, TGF-β growth inhibition, or hypermotility on Lam322′. Distinct from its role as a mitogen, EGF proved to be a required cofactor for TGF-β-induced hypermotility and could not be replaced by HGF or KGF. Prostate epithelial cells have a short replicative lifespan, restricted both by p16^INK4A and telomere-related mechanisms. We immortalized the normal prostate epithelial cell line HPrE-1 by transduction to express bmi1 and TERT. Prostate epithelial cells lose expression of p63, β4 integrin, and Lam332 when they transform to invasive carcinoma. In contrast, HPrE-1/bmi1/TERT cells retained expression of these proteins and normal TGF-β signaling and hypermotility for >100 doublings. Thus, keratinocytes and prostate epithelial cells possess common hypermotility and senescence mechanisms and immortalized prostate cell lines can be engineered using defined methods to yield cells retaining normal properties.     

Interleukin-10 in serous ovarian carcinoma cell lines

 Interleukin-10, one of the most potent anti-inflammatory cytokines, is expressed in ovarian carcinomas in vivo. In contrast to the high levels of IL-10 in ascites and tumour tissue, the expression of this cytokine appears to be a rare event in ovarian carcinoma cell lines in vitro. Virtually nothing is known about the regulation of IL-10 expression in ovarian carcinoma cell lines. We investigated the expression of IL-10 in four cell lines originally derived from ovarian serous adenocarcinoma: OVCAR-3, SKOV-3, CAOV-3 and OAW-42. IL-10-specific mRNA was detected in OVCAR-3 and only this cell line produced IL-10 constitutively under serum-free conditions as well as in serum-containing medium. Our studies on the regulation of IL-10 secretion in OVCAR-3 revealed that (1) proinflammatory stimuli IL-1β and TNF-α, but not LPS, enhance IL-10 secretion, (2) IL-6 has no influence on the release of IL-10, (3) prostaglandin E2 influences neither the spontaneous nor the TNF-α- or IL-1β-stimulated IL-10 production and (4) interferon-γ inhibits IL-10 secretion. We conclude that only a minority of serous ovarian carcinoma cells maintain the ability to produce IL-10 in vitro. Our data on the regulation of IL-10 production in OVCAR-3 indicate that ovarian carcinoma cells share some, but not all, of the regulatory features typical for the monocytic IL-10 secretion. Received: 1 February 2001 / Accepted: 29 March 2001     

<Emphasis Type="Italic">Scutellaria</Emphasis> extract and wogonin inhibit tumor-mediated induction of T<Subscript>reg</Subscript> cells via inhibition of TGF-β1 activity

A number of studies have implicated tumor-induced T_reg cell activity in the sub-optimal response to therapeutic vaccines. Development of neo-adjuvant strategies targeting T_reg cells is therefore imperative. Scutellaria extracts or constituent flavonoids have shown encouraging efficacy against various tumors, including gliomas, in both pre-clinical and clinical studies. We report here, for the first time, that Scutellaria ocmulgee leaf extract (SocL) and flavonoid wogonin could inhibit TGF-β1-induced T_reg activity in malignant gliomas. F344 rats, subcutaneously transplanted with F98 gliomas, were treated with SocL. There was a significant inhibition of intra-tumoral TGF-β1 and T_reg cell frequency as well as peripheral blood TGF-β1 levels in SocL-treated animals compared to the controls. SocL extract and wogonin also inhibited glioma-induced, TGF-β1-mediated T_reg activity in vitro. SocL extract and wogonin also inhibited the secretion of IL-10 in T_reg culture; whereas the level of IL-2 was either unchanged or marginally enhanced. We also observed an inhibition of Smad-3, GSK-3β and ERK1/2 signaling by SocL and wogonin in T_reg cells, while phosphorylation of P38 MAPK was considerably enhanced, indicating that SocL or wogonin could inhibit the T cells’ response to TGF-β1 via modulation of both Smad and non-Smad signaling pathways. Overall, this study suggests that Scutellaria can potentially reverse tumor-mediated immune suppression via inhibition of TGF-β1 secretion as well as via inhibition of T cells’ response to TGF-β1. This may provide an opportunity for developing a novel adjuvant therapeutic strategy for malignant gliomas, combining Scutellaria with immunotherapy and chemo/radio-therapeutic regimen, which could potentially improve the disease outcome.