Nuclear factor kappa B plays a pivotal role in polyinosinic-polycytidylic acid-induced expression of human ß-defensin 2 in intestinal epithelial cells.

Intestinal epithelial cells (IECs) play an important role in protecting the intestinal surface from invading pathogens by producing effector molecules. IECs are one of the major sources of human beta-defensin 2 (hBD-2), and can produce it in response to a variety of stimuli. Although IECs express Toll-like receptor 3 (TLR-3) and can respond to its ligand, double-stranded RNA (dsRNA), hBD-2 expression in response to dsRNA has not been elucidated. In the present study, using an artificial analogue of dsRNA, polyinosinic-polycytidylic acid (poly I:C), we investigated whether the human IEC line, HT-29, can produce hBD-2 in response to poly I:C. HT-29 cells can express hBD-2 mRNA only when stimulated with poly I:C. The induction of hBD-2 mRNA expression was observed at 3 h after stimulation and peaked at 12 h of post-stimulation. Pre-incubation of the cells with nuclear factor kappa B (NF-κB)-specific inhibitor, l-1-4'-tosylamino-phenylethyl-chloromethyl ketone (TPCK) and isohelenine abolished the expression of hBD-2. Detection of the poly I:C signal by TLR-3 on the surface of HT-29 cells was revealed by pre-incubating the cells with anti-TLR-3 antibody. The 5'-regulatory region of the hBD-2 gene contains two NF-κB binding sites. A luciferase assay revealed the importance of the proximal NF-κB binding site for poly I:C-induced expression of hBD-2. Among NF-κB subunits, p65 and p50 were activated by poly I:C stimulation and accumulated in the nucleus. Activation of the p65 subunit was investigated further by determining its phosphorylation status, which revealed that poly I:C stimulation resulted in prolonged phosphorylation of p65. These results indicate clearly that NF-κB plays an indispensable role in poly I:C induced hBD-2 expression in HT-29 cells.

Tumour necrosis factor-alpha-mediated human polymeric immunoglobulin receptor expression is regulated by both mitogen-activated protein kinase and phosphatidylinositol-3-kinase in HT-29 cell line.

Human polymeric immunoglobulin receptor (pIgR) is present on the surface of glandular epithelium, and it plays a crucial role in the mucosal immune defence. pIgR expression in HT-29 cells is up-regulated by one of the proinflammatory cytokines, tumour necrosis factor (TNF)-alpha. However, the mechanism used by the TNF-alpha-mediated signalling pathway has not been examined exclusively. To elucidate this mechanism in detail, HT-29 cells were cotreated with TNF-alpha and mitogen-activated protein kinase kinase (MAPKK, also called MEK1) inhibitor, PD98059, and the amount of free secretory component (SC) secreted into the culture medium was measured. The amount of free SC stimulated by TNF-alpha was increased by addition of PD98059. This up-regulation occurred at the transcriptional level. The amount of SC was also up-regulated by addition of TNF-alpha with U0126, an inhibitor of MEK1 and MEK2. Nuclear factor (NF)-kappaB activity and NF-kappaB binding to the kappaB2 site localized upstream of the pIgR gene did not change after coincubation of HT-29 cells with TNF-alpha and PD98059. The expression level of pIgR by TNF-alpha was decreased by LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3K), at the transcriptional level. Extracellular signal-regulated kinase (ERK)1/2 phosphorylation and NF-kappaB binding to the kappaB2 site were not affected by LY294002 treatment. These data suggest that TNF-alpha-mediated pIgR expression is negatively regulated by ERK pathway, which is independent of NF-kappaB. In addition, decrease of SC production by Ly294002 suggests that the presence of PI3K mediated regulation of SC production.

Dietary fructooligosaccharides up-regulate immunoglobulin A response and polymeric immunoglobulin receptor expression in intestines of infant mice.

We examined whether or not dietary fructooligosaccharides (FOS) in infancy can have a beneficial effect on the mucosal immune system. Newborn BALB/c mice, accompanied by their dams until 21 days of age, were fed either a control diet based on casein [FOS- diet group] or a FOS- diet supplemented with 5% (w/w) FOS [FOS+ diet group]. Total IgA levels in tissue extracts from the intestines of mice in the FOS+ diet group at 38 days of age were about twofold higher (P < 0.05) than those in the FOS- diet group in the jejunum, ileum and colon. Ileal and colonic polymeric immunoglobulin receptor (pIgR) expression in the FOS+ diet group at 36 days of age was 1.5-fold higher than in the FOS- diet group (P < 0.05). Consistent with these results, the ileal IgA secretion rate of the FOS+ diet group at 37 days of age was twofold higher than that of the FOS- diet group (P < 0.05). Moreover, the percentage of B220(+)IgA+ cells in Peyer's patches (PP) was significantly higher in the FOS+ diet group than in the FOS- diet group (6.2%versus 4.3%, P < 0.05), suggesting that isotype switching from IgM to IgA in PP B cells might be enhanced in vivo. Taken together, our findings suggest that dietary FOS increases the intestinal IgA response and pIgR expression in the small intestine as well as the colon in infant mice.

Retinoic acid enhances the gene expression of human polymeric immunoglobulin receptor (pIgR) by TNF-alpha.

In this study, the detailed mechanisms for the effects of vitamin A on the expression of polymeric immunoglobulin receptor (pIgR) were examined. Expression of the pIgR by tumour necrosis factor (TNF-alpha) was enhanced by the addition of all-trans retinoic acid (ATRA) or 9-cis retinoic acid (9CRA). This enhancement was mediated mainly by RARalpha, and regulated at the transcriptional level. Transcription factor nuclear factor-kappaB (NF-kappaB) binding and activation were not influenced by addition of ATRA. These data imply that RA, in combination with TNF-alpha, could up-regulate the expression of pIgR. In addition, we hypothesize that up-regulation of pIgR by RA is controlled through the RAR-dependent signalling pathway and that it plays a role in enhancement of mucosal immunity.

Release of non-glycosylated polymeric immunoglobulin receptor protein.

Using a recombinant vaccinia virus containing the T7 RNA polymerase, we have established a system for the transient expression of human polymeric immunoglobulin receptor (pIgR) in baby hamster kidney cells, a baby hamster-derived fibroblastic cell line. This transfection system resulted in the successful expression of pIgR in these cells, and Western blot analysis showed that human pIgR was expressed as two different molecular weight forms of 92 and 107 kDa. Treatment with endoglycosidase H showed that the difference between these two forms was due to the glycosylation status of the protein. In order to examine the functional role of glycosylation, we treated the transfected cells with tunicamycin, which prevents a core glycosylation step in the endoplasmic reticulum. Non-glycosylated pIgR was released into the culture medium of the transfected cells, albeit with extremely low efficiency. Taking these results together, we conclude that the glycosylation of pIgR may play a positive role in the efficient transport or release of free pIgR.

Role of nuclear factor-kappaB in the expression by tumor necrosis factor-alpha of the human polymeric immunoglobulin receptor (plgR) gene.

We analyzed the mechanism of human polymeric immunoglobulin receptor (pIgR) gene upregulation by tumor necrosis factor (TNF)-alpha. Northern blot analysis showed that the expression of pIgR mRNA was enhanced by TNF-alpha stimulation. This activation was completely inhibited by RNA polymerase or protein synthesis inhibitors, suggesting that the regulation of pIgR gene expression depends on de novo RNA and protein synthesis. Furthermore, the stimulation of pIgR mRNA by TNF-alpha was decreased by pyrrolidinedithiocarbamate and L-1-4'-tosylamino-phenylethyl-chloromethyl ketone, which are known nuclear factor (NF)-kappaB inhibitors. For further analysis of gene regulation, we cloned and sequenced the 1.5-kb 5'-flanking region of the pIgR gene. In the upstream region, we found two NF-kappaB-binding motifs (named kappaB1 and kappaB2 from the 5' region). An electrophoretic mobility shift assay indicated that two components of the NF-kappaB/Re1 family, p50 and p65, bound with higher affinity to the KB2 element than to the kappaB1 element. We also analyzed plgR gene expression using reporter plasmids expressing the firefly luciferase gene. Stimulation by TNF-alpha significantly activated the pIgR gene promoter, as a 775-bp upstream region of the pIgR gene increased luciferase gene expression in cells treated with TNF-alpha. The activation of promoter activity by TNF-alpha was abolished when a mutation was inserted into kappaB1 or kappaB2. These data indicated that pIgR gene expression induced by TNF-alpha is transcriptionally regulated via activation of NF-kappaB. In addition, there is a possibility that another factor may act in concert with NF-kappaB.