Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.

The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.

Psg18 is specifically expressed in follicle-associated epithelium.

Pregnancy-specific glycoproteins (Psgs) secreted by the placenta regulate the immune system to ensure the survival of the fetal allograft by inducing IL-10, an anti-inflammatory cytokine. However, it is unknown whether Psgs are involved in more general aspects of immune response other than maternal immunity. Here, we report that Psg18 is highly expressed in the follicle-associated epithelium (FAE) overlaying Peyer's patches (PPs). Bioinformatics analysis with Reference Database for Immune Cells (RefDIC) as well as RT-PCR data demonstrated that Psg18 is exclusively expressed in FAE in adult mice, in contrast to other Psg family members that are either not expressed or only slightly expressed in FAE. Psg18 expression was observed in FAE of germ-free-conditioned mice, and was slightly upregulated after bacterial inoculation. In situ hybridization analysis revealed that Psg18 is widely expressed throughout FAE. Furthermore, Psg18 protein is deposited on the extracellular matrix in the subepithelial dome beneath FAE, where antigen-presenting cells accumulate. These results suggest that Psg18 is an FAE-specific marker protein that could promote interplay between FAE and immune cells in mucosa-associated lymphoid tissues.

Distinct gene expression profiles characterize cellular phenotypes of follicle-associated epithelium and M cells.

Follicle-associated epithelium (FAE) covering Peyer's patches contains specialized epithelial M cells that take up ingested macromolecules and microorganisms from the lumen of the gut by transcytosis. Using high-density oligonucleotide microarrays, we analyzed the gene expression profiles of FAE and M cells in order to characterize their cellular phenotypes. The microarray data revealed that, among approximately 14,000 genes, 409 were expressed in FAE at twofold or higher levels compared to the intestinal epithelial cells (IECs) of the villi. These included genes involved in membrane traffic, host defense and transcriptional regulation, as well as uncharacterized genes. Real-time PCR and in situ hybridization analyses identified three molecules, ubiquitin D (Ub-D), tumor necrosis factor receptor superfamily 12a (TNFRsf12a), and transmembrane 4 superfamily 4 (Tm4sf4), which were predominantly distributed throughout FAE, but were expressed little, if at all, in IECs. By contrast, transcripts of secretory granule neuroendocrine protein 1 (Sgne-1) were scattered in FAE, and were co-localized with Ulex europaeus agglutinin-1 (UEA-1)-positive cells. This clearly suggests that expression of Sgne-1 in the gut is specific to M cells. Such a unique pattern of gene expression distinguishes FAE and M cells from IECs, and may reflect their cellular phenotype(s) associated with specific functional features.

Recovery from heat shock injury by activation of Na+-glucose cotransporter in renal epithelial cells.

Exposure of cells or organs to sublethal physical or chemical stresses induces disruption of cellular structures and functions. Here, we examined whether Na(+)-glucose cotransporter (SGLT1) is involved in the recovery from heat shock (HS) injury in porcine renal epithelial LLC-PK(1) cells. Recovery from HS (42 degrees C for 3 h, then 37 degrees C for 12 h) increased SGLT1 activity, assessed by [14C]alpha-methyl glucopyranoside uptake, and a maximal transport rate (V(max)) from 2.4 to 5.9 nmol/mg protein/30 min, but did not alter an apparent affinity constant (K(m)). Protein distribution of SGLT1 in apical membrane fraction was also increased after recovery from HS without changing in total membrane fraction. Membrane integrity assessed by calcein accumulation was decreased by HS, and then returned to basal level. This recovery was inhibited by phloridzin, a potent SGLT1 inhibitor, and nonmetabolizable glucose analogues. Anti-transforming growth factor-beta 1 (TGF-beta 1) antibody inhibited both elevation of SGLT1 distribution in apical membrane and recovery of calcein accumulation induced by HS. Taken together, HS increases in the number of SGLT1 protein in apical membrane mediated via TGF-beta 1 signaling pathway. The increase of glucose uptake is necessary to repair plasma membrane integrity.

Up-regulation of sodium-dependent glucose transporter by interaction with heat shock protein 70.

Heat shock stress induces some heat shock proteins, including Hsp70, and activates sodium-dependent glucose transport in porcine renal LLC-PK(1) cells, but its mechanisms have not been described in detail. We investigated whether sodium-dependent glucose transporter (SGLT1) interacts with Hsp70 to increase SGLT1 activity. Heat shock stress increased SGLT1 activity without changing SGLT1 expression. The increase of SGLT1 activity was completely inhibited by an anti-transforming growth factor-beta1 (TGF-beta1) antibody. Instead of heat shock stress, TGF-beta1 increased SGLT1 activity dose- and time-dependently without changing SGLT1 expression. We found that the amount of Hsp70 immunoprecipitated from TGF-beta1-treated cells with an anti-SGLT1 antibody was higher than that of the control cells. Transfection of an anti-Hsp70 antibody into the cells inhibited the increase of SGLT1 activity. With confocal laser microscopy, both SGLT1 and Hsp70 was localized near the apical membrane in the TGF-beta1-treated cells, and an anti-Hsp70 antibody disturbed this localization. Furthermore, we clarified that an anti-Hsp70 antibody inhibited interaction of SGLT1 with Hsp70 in vitro. These results suggest that Hsp70 forms a complex with SGLT1 and increases the expression level of SGLT1 in the apical membrane, resulting in up-regulation of glucose uptake.

Phosphatidylinositol 3-kinase mediates inhibitory effect of angiotensin II on sodium/glucose cotransporter in renal epithelial cells.

Effects of angiotensin II (ANGII) on regulation of sodium/glucose cotransporter (SGLT1) activity were investigated in LLC-PK(1) cells, renal proximal epithelial cell line. ANGII inhibited alpha-[14C] methyl-D-glucopyranoside (AMG) uptake into LLC-PK(1) cells in a dose-dependent manner. This inhibition was based on a decrease in maximal transport rate (Vmax) of AMG from 2.20 nmol/mg protein/15 min to 1.19 nmol/mg protein/15 min, although apparent affinity constant (Km) did not alter. In western blot analysis, protein level of SGLT1 in brush border membrane (BBM) was decreased by ANGII, although total SGLT1 was not altered. In the aspect of intracellular signal transduction, ANGII blocked the formation of cAMP. Pertussis toxin, an inactivator of Gi protein that control intracellular cAMP level, completely prevented the decrease of AMG uptake caused by ANGII. 8-Br-cAMP, a cell membrane permeable cAMP analogue, increased AMG uptake and protein level of SGLT1 in BBM. Both wortmannin and LY294002 that are phosphatidylinositol (PI) 3-kinase inhibitors, inhibited the SGLT1 activity, and also attenuated the effect of 8-Br-cAMP on SGLT1 activity. Those inhibitors prevented the 8-Br-cAMP-induced expression of SGLT1 in plasma membrane. We conclude that ANGII plays an important role in post-translational regulation in SGLT1. Inhibition of SGLT1 translocation is suggested to be caused by inactivation of protein kinase A and decrease of PI 3-kinase activity.