Glutamine decreases lipopolysaccharide-induced IL-8 production in Caco-2 cells through a non-NF-kappaB p50 mechanism

Glutamine (Gln) supplementation has been shown to decrease production of pro-inflammatory cytokines by the human intestinal mucosa. The mechanism of this is poorly understood. We hypothesize that Gln down-regulates lipopolysaccharide (LPS)-stimulated pro-inflammatory cytokine production in Caco-2 cells by nuclear factor-kappa B (NF-kappaB). Caco-2 cells were incubated with different concentrations of Gln with or without methionine sulfoximine (MS, an inhibitor of glutamine synthetase) before stimulation with LPS. IL-6, IL-8, IL-10 and TNF-alpha protein and mRNA level were determined. NF-kappaB translocation was determined using an ELISA-based kit. IL-8 was the only detectable cytokine/chemokine. The largest amount of IL-8 was secreted by cells in the presence of MS with no Gln in the medium after exposure to LPS. LPS increased IL-8 production, peaking 10h after LPS administration. The addition of Gln (0.5 or 5.0mM) decreased IL-8 peptide and mRNA expression. LPS increased NF-kappaB nuclear translocation in the presence or absence of MS. Neither Gln nor MS altered NF-kappaB nuclear translocation. These results indicate that the lack of glutamine increases IL-8 production by Caco-2 cells after LPS stimulation. However, the glutamine-mediated decrease in LPS-stimulated IL-8 production is not associated with NF-kappaB p50 nuclear binding.     

Statins directly suppress cytokine production in murine intraepithelial lymphocytes

Statins, inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are known not only as cholesterol-lowering agents but also as anti-inflammatory mediators. However, their regulatory effect on intestinal mucosal immunity remains unclear. The present study examined the possible direct effects of statin on intestinal intraepithelial lymphocytes (IELs), the front line cells of the intestinal mucosal immune system. Murine IELs were isolated from the small intestines of C57BL/6 mice. IELs activated with anti-CD3/CD28 monoclonal antibodies produced interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and IL-4 in significant numbers; however, they did not produce IL-5. Both simvastatin and lovastatin suppressed IEL production of IFN-γ, TNF-α, IL-2, and IL-4 in a dose-dependent manner, whereas 48-h treatment with high concentrations (5 × 10^?5 M) of simvastatin and lovastatin did not affect the number of IELs. The suppressive effect of the simvastatin was significantly restored by the addition of mevalonate, farnesyl pyrophosphate ammonium salt, and geranylgeranyl pyrophosphate ammonium salt, which are downstream metabolites of HMG-CoA. These findings suggest that statins have direct suppressive effects on the production of T helper 1-cytokines and IL-4 in IELs; these effects are associated with inhibition of the mevalonate pathway to some extent.     

Effects of glutamine supplementation on the immune status in weaning piglets with intrauterine growth retardation

Neonates with intrauterine growth retardation (IUGR) often suffer from impaired cellular immunity, and weaning may further aggravate adverse effects of IUGR on development and function of the immune system. In this study, we investigated effects of glutamine supplementation on immune status in the intestines of weaning pigs with IUGR, focusing on molecular mechanisms underlying altered immune response. Piglets with IUGR were weaned at 21 days of age and received orally 1.22 g alanine or 1 g glutamine per kg body weight every 12?h. Weight gain and intestinal weight of weaning piglets were increased by glutamine supplementation. Levels of serum IgG in piglets supplemented with glutamine were increased compared with Control piglets. The production of IL-1 and IL-8 in the serum and jejunum was decreased by glutamine supplementation, whereas the levels of IL-4 in the serum and the concentrations of IL-4 and IL-10 in the jejunum were increased. The expression of heat shock protein 70 (Hsp70) in the jejunum was increased by glutamine supplementation, but the degradation of inhibitor?κB and the activity of nuclear factor-κB (NF-κB) were decreased. In conclusion, glutamine supplementation enhanced immune response in weaning piglets with IUGR. The effects of glutamine in IUGR are associated with increased Hsp70 expression and suppression of NF-κB activation.     

PPARγ inhibits airway epithelial cell inflammatory response through a MUC1-dependent mechanism

This study was conducted to examine the relationship between the peroxisome proliferator-associated receptor-γ (PPARγ) and MUC1 mucin, two anti-inflammatory molecules expressed in the airways. Treatment of A549 lung epithelial cells or primary mouse tracheal surface epithelial (MTSE) cells with phorbol 12-myristate 13-acetate (PMA) increased the levels of tumor necrosis factor (TNF)-α in cell culture media compared with cells treated with vehicle alone. Overexpression of MUC1 in A549 cells decreased PMA-stimulated TNF-α levels, whereas deficiency of Muc1 expression in MTSE cells from Muc1 null mice increased PMA-induced TNF-α levels. Treatment of A549 or MTSE cells with the PPARγ agonist troglitazone (TGN) blocked the ability of PMA to stimulate TNF-α levels. However, the effect of TGN required the presence of MUC1/Muc1, since no differences in TNF-α levels were seen between PMA and PMA plus TGN in MUC1/Muc1-deficient cells. Similarly, whereas TGN decreased interleukin-8 (IL-8) levels in culture media of MUC1-expressing A549 cells treated with Pseudomonas aeruginosa strain K (PAK), no differences in IL-8 levels were seen between PAK and PAK plus TGN in MUC1-nonexpressing cells. EMSA confirmed the presence of a PPARγ-binding element in the MUC1 gene promoter. Finally, TGN treatment of A549 cells increased MUC1 promoter activity measured using a MUC1-luciferase reporter gene, augmented MUC1 mRNA levels by quantitative RT-PCR, and enhanced MUC1 protein expression by Western blot analysis. These combined data are consistent with the hypothesis that PPARγ stimulates MUC1/Muc1 expression, thereby blocking PMA/PAK-induced TNF-α/IL-8 production by airway epithelial cells.     

Intestinal intraepithelial lymphocyte gamma delta-T cell-derived keratinocyte growth factor modulates epithelial growth in the mouse

Keratinocyte growth factor (KGF) promotes intestinal epithelial growth. To understand the relevance of intraepithelial lymphocyte (IEL)-derived KGF expression on epithelial growth, we used a mouse model of villus atrophy by the administration of total parenteral nutrition, and a model of villus hypertrophy by the creation of a short bowel syndrome. KGF expression was confined to gammadelta-TCR(+) IELs. IEL-derived KGF expression was highest in the crypts, somewhat less in the lower portion of villi, and markedly lower in the upper portion of villi. Total parenteral nutrition administration was associated with a down-regulation of IEL-derived KGF expression, and short bowel syndrome was associated with an up-regulation of IEL-derived KGF expression. In the absence of gammadelta-TCR(+) IEL, using gammadelta(-/-) mice, intestinal epithelial cell proliferation decreased in control, and in both mucosal atrophy (22% decline) and mucosal hypertrophy (14%) models. These results show that KGF from IELs is an important factor for maintenance of intestinal epithelial cell proliferation and villus growth.     

Preventive oral supplementation with glutamine and arginine has beneficial effects on the intestinal mucosa and inflammatory cytokines in endotoxemic rats

The objective of this study was to evaluate the effect of oral supplementation with a combination of arginine and glutamine on the intestinal mucosa and inflammatory cytokines of lipopolysaccharide (LPS)-induced adult rats. Fifty Sprague-Dawley rats (average weight of 185 ± 15 g) were randomly divided into five groups: control group A (CA) and control group B (CB), both orally supplemented with 0.9% saline; group Arg, supplemented with 300 mg/kg day(-1) arginine; group Gln, supplemented with 300 mg/kg day(-1) glutamine; group AG, supplemented with 150 mg/kg day(-1) arginine and 150 mg/kg day(-1) glutamine. The experiment lasted for 2 weeks. Food intake and body weight were measured during the experiment. At 10.00 h of day 15, animals were injected with 4 mg/kg LPS (group CB, Arg, Gln, and AG) or sterile saline (group CA) after supplementation. Then at 14.00 h, all animals were killed and blood and tissue collected. The results showed that compared with group CB, arginine concentration tended to be increased (P > 0.05) in group Arg and AG, while there was no significant difference in glutamine concentration among the groups challenged with LPS. Oral supplementation with arginine or/and glutamine mitigated morphology impairment (lower villus height, P < 0.05) in the jejunum and ileum induced by LPS challenge. LPS administration resulted in a significant increase in TNF-α, IL-1β, IL-6 and IL-10 mRNA abundance. Arginine only significantly decreased TNF-α mRNA abundance in the ileum, while glutamine significantly decreased both TNF-α and IL-10 mRNA in the ileum. A combination of arginine and glutamine significantly decreased TNF-α and IL-1β mRNA abundance in both the jejunum and ileum, while they also significantly decreased anti-inflammatory IL-10 in the ileum. These results revealed that an oral supply of combined arginine and glutamine had more favorable effects on the intestinal mucosa and inflammatory cytokines than a supply of arginine or glutamine alone.     

Effects of rhinovirus infection on histamine and cytokine production by cell lines from human mast cells and basophils

To understand the biochemical events that occur in the airways after rhinovirus (RV) infection, we developed for the first time a model in which the cell lines from human mast cells (HMC-1) and basophils (KU812) can be infected with RV14, a major group RV. Viral infection was confirmed by demonstrating that viral titers in culture supernatants, and RV RNA increased with time. RV14 infection alone and a combination of PMA plus calcium ionophore A23187, did not increase histamine production by these cells, although IgE plus anti-IgE increased the histamine production. However, histamine content in the supernatants increased in response to PMA plus A23187, or IgE plus anti-IgE after RV14 infection. PMA plus A23187 or IgE plus anti-IgE induced the production of IL-8 and GM-CSF in supernatants of HMC-1 cells and IL-4 and IL-6 in supernatants of KU812 cells. RV14 infection further increased the production of the cytokines, whereas RV14 infection alone did not alter the production of the cytokines by these cells. An Ab to ICAM-1 inhibited RV14 infection of the cells and decreased the production of cytokines and histamine after RV14 infection. RV14 infection enhanced the increases in intracellular calcium concentration and activation of NF-kappaB by PMA plus A23187 in the cells. These findings suggest that RV14 infection may prime the cytokine and histamine production from mast cells and basophils and may cause airway inflammation in asthma.     

Decline in intestinal mucosal IL-10 expression and decreased intestinal barrier function in a mouse model of total parenteral nutrition

Loss of intestinal epithelial barrier function (EBF) is a major problem associated with total parenteral nutrition (TPN) administration. We have previously identified intestinal intraepithelial lymphocyte (IEL)-derived interferon-gamma (IFN-gamma) as a contributing factor to this barrier loss. The objective was to determine whether other IEL-derived cytokines may also contribute to intestinal epithelial barrier breakdown. C57BL6J male mice received TPN or enteral nutrition (control) for 7 days. IEL-derived interleukin-10 (IL-10) was then measured. A significant decline in IEL-derived IL-10 expression was seen with TPN administration, a cytokine that has been shown in vitro to maintain tight junction integrity. We hypothesized that this change in IEL-derived IL-10 expression could contribute to TPN-associated barrier loss. An additional group of mice was given exogenous recombinant IL-10. Ussing chamber experiments showed that EBF markedly declined in the TPN group. TPN resulted in a significant decrease of IEL-derived IL-10 expression. The expression of several tight junction molecules also decreased with TPN administration. Exogenous IL-10 administration in TPN mice significantly attenuated the TPN-associated decline in zonula occludens (ZO)-1, E-cadherin, and occludin expression, as well as a loss of intestinal barrier function. TPN administration led to a marked decline in IEL-derived IL-10 expression. This decline was coincident with a loss of intestinal EBF. As the decline was partially attenuated with the administration of exogenous IL-10, our findings suggest that loss of IL-10 may be a contributing mechanism to TPN-associated epithelial barrier loss.     

Induction of interferon gamma in human gingival fibroblasts challenged with phytohaemagglutinin

Interferon gamma (IFN-gamma) is a potential immunoregulatory cytokine, which is secreted mainly by cells of immune origin. In this study, we examined the capacity of human gingival fibroblasts as non-professional immune cells to express IFN-gamma messenger RNA (mRNA) and to produce the protein. Cultures of fibroblast cells were established from gingival biopsies from three children. The expression of mRNA for IFN-gamma was studied by in situ hybridization, and the level of IFN-gamma was determined by cell-released capturing ELISA. Treatment of the cells with phytohaemagglutinin (PHA) (2.5, 5.0, and 10 microg/ml) increased the number of IFN-gamma mRNA expressing cells and the protein production at 1, 6, and 24 h. Non-stimulated cells did not reveal measurable levels of IFN-gamma mRNA or the protein. The inflammatory cytokines interleukin 1beta (IL-1beta) (100 microg/ml) and tumour necrosis factor alpha (TNFalpha) (10 ng/ml) did not affect IFN-gamma mRNA expression or protein production. Treatment of the cells with 1 microM phorbol 12-myristate-13-acetate (PMA) stimulated IFN-gamma mRNA expression but had no effect on IFN-gamma protein production. We conclude that human gingival fibroblasts not only transcribe IFN-gamma mRNA but also produce the IFN-gamma protein in response to PHA. The finding that human gingival fibroblasts, produce the cytokine IFN-gamma, further support the concept that these cells take an active part in the modulation of the inflammatory and immune response in the periodontal tissue.     

Multiple levels of activation of murine CD8(+) intraepithelial lymphocytes defined by OX40 (CD134) expression: effects on cell-mediated cytotoxicity, IFN-gamma, and IL-10 regulation.

The involvement of OX40 (CD134) in the activation of CD8(+) intestinal intraepithelial lymphocytes (IELs) has been studied using freshly isolated IELs and in vitro CD3-stimulated IELs. Although freshly isolated CD8(+) IELs exhibited properties of activated T cells (CD69 expression and ex vivo cytotoxicity), virtually all CD8(+) IELs from normal mice were devoid of other activation-associated properties, including a lack of expression of OX40 and the ligand for OX40 (OX40L) and an absence of intracellular IFN-gamma staining. However, OX40 and OX40L expression were rapidly up-regulated on CD8 IELs following CD3 stimulation, indicating that both markers on IELs reflect activation-dependent events. Unlike IELs, activated lymph node T cells did not express OX40L, thus indicating that OX40-OX40L communication in the intestinal epithelium is part of a novel CD8 network. Functionally, OX40 expression was exclusively associated with IELs with active intracellular IFN-gamma synthesis and markedly enhanced cell-mediated cytotoxicity. However, OX40 costimulation during CD3-mediated activation significantly suppressed IL-10 synthesis by IELs, whereas blockade of OX40-OX40L by anti-OX40L mAb markedly increased IL-10 production. These findings indicate that: 1) resident CD69(+)OX40(-) IELs constitute a population of partially activated T cells poised for rapid delivery of effector activity, 2) OX40 and OX40L expression defines IELs that have undergone recent immune activation, 3) OX40(+) IELs are significantly more efficient CTL than are OX40(-) IELs, and 4) the local OX40/OX40L system plays a critical role in regulating the magnitude of cytokine responses in the gut epithelium.     

Autocrine IL-15 mediates intestinal epithelial cell death via the activation of neighboring intraepithelial NK cells

Intestinal intraepithelial lymphocytes (IELs), which reside between the basolateral faces of intestinal epithelial cells (IECs), provide a first-line defense against pathogens via their cytotoxic activity. Although IEC-derived IL-7 and IL-15 are key regulatory cytokines for the development and activation of IELs, we report here that IL-15 but not IL-7 mediates the reciprocal interaction between IELs and IECs, an important interaction for the regulation of appropriate mucosal immunohomeostasis. IL-15-treated IELs induced cell death in IECs via the cytotoxic activity in vitro. Among the different subsets of IL-15-treated IELs, CD4(-)CD8(-)TCR(-) IELs, which express NK marker (DX5 or NK1.1), showed the most potent syngenic IEC killing activity. These intraepithelial NK cells expressed Ly-49 molecules, NKG2 receptors, and perforin. These results suggest the possibility that the cell death program of IECs could be regulated by self-produced IL-15 through the activation of intraepithelial NK cells.     

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.     

T-deficient transmembrane signaling in CD4+ T cells of retroviral-induced immune-deficient mice.

The defective virus found in the LP-BM5 mixture of murine leukemia viruses induces a severe immune deficiency disease in C57BL/6 mice that is characterized by the activation and expansion of T and B cells that become unresponsive to normal immune stimuli. The nature of the biochemical lesion in these defective lymphocyte populations remains unknown. Flow cytometric analysis of the T cell population in infected animals has demonstrated expansion of both CD4+ and CD8+ subsets. Despite chronic expansion in vivo, CD4+ T cells by wk 4 postinfection failed to up-regulate cell surface IL-2R expression, produced IL-2, or proliferate in vitro in response to either Con A, Staphylococcal enterotoxin super-antigens, or anti-CD3 stimulation. Exogenous IL-2 did not restore the proliferative response and also failed to up-regulate IL-R expression on CD4+ T cells from infected mice, even though basal IL-2R expression was initially elevated compared to normals. In contrast, CD4+ T cells from infected mice could be induced to proliferate by stimulation with PMA and ionomycin resulting in IL-2R up-regulation, IL-2 production, and proliferation. Moreover, proliferation could also be induced by anti-CD3 plus PMA, although anti-CD3 plus ionomycin was without effect. These studies suggest that chronic expansion of CD4+ T cells in infected mice is probably not maintained by normal TCR signaling, which appears defective in these cells. In addition, the lesion in biochemical signaling appears to result in defective activation of protein kinase C, which can be overcome by direct activation with PMA.     

Allogeneic non-T spleen cells restore the responsiveness of normal T cell clones stimulated with antigen and chemically modified antigen-presenting cells

Stimulation of IL-2-producing T cell clones with chemically modified APC and Ag induces a state of proliferative unresponsiveness, i.e., subsequent stimulation with normal APC and Ag fails to elicit IL-2 production. One possible effect of chemical modification on the APC is the destruction of its ability to provide costimulatory signals. To test this, various potential costimulators were added to T cells at the time of exposure to Ag and chemically modified APC. None of the cytokines tested, including IL-1, had a positive effect; however, addition of allogeneic spleen cells allowed a T cell proliferative response and prevented the induction of subsequent unresponsiveness. Fractionation of the spleen cells showed that low density B cells and macrophages were the best source of costimulatory activity. Allogeneic resting B cells provided some costimulatory activity and resting T cells, none at all. Attempts to mimic costimulatory signals with the phorbol ester PMA were only partially successful. PMA prevented the induction of T cell unresponsiveness but failed to allow T cell proliferation in response to Ag plus chemically modified APC. Our results suggest that IL-2 production by normal T cell clones is dependent not only on T cell receptor occupancy, but also on short range costimulatory signals that are provided to different degrees by various non-T accessory cells.     

Hepatocyte growth factor and keratinocyte growth factor enhance IL-1-induced IL-8 secretion through different mechanisms in Caco-2 epithelial cells

A variety of cytokines have been detected in inflamed intestinal mucosal tissues, including the pro-inflammatory cytokine, interleukin-1 (IL-1), along with growth factors involved in wound healing processes such as proliferation and cell migration. However, little is known about how IL-1 and growth factors interact with intestinal epithelial cells to regulate the production of inflammatory cytokines such as interleukin-8 (IL-8). Previously, we have shown that hepatocyte growth factor (HGF) could significantly enhance IL-1-stimulated IL-8 secretion by the Caco-2 colonic epithelial cell line, yet HGF, by itself, did not stimulate IL-8 secretion. In this report, a second growth factor, keratinocyte growth factor (KGF), was also found to significantly enhance IL-1-induced IL-8 secretion by Caco-2 cells, yet KGF, by itself, also had no effect. Simultaneous addition of both IL-1 and KGF was also required for the enhancing effect. Treatment of the Caco-2 cells with wortmannin or triciribine suppressed the enhancing effect of HGF, suggesting that the effect was mediated by signaling through phosphatidylinositol-3-kinase (PI3K) and the kinase AKT. The enhancing effect of KGF was not affected by wortmannin, but was suppressed by triciribine, suggesting that the effect of KGF was through a PI3K-independent activation of AKT. These results suggest that the growth factors HGF and KGF may play a role in enhancing IL-1-stimulated production of IL-8 by epithelial cells during mucosal inflammations. However, the mechanism by which the growth factors enhance the IL-1 response may be through different initial signaling pathways.