Ligation of intestinal epithelial CD1d induces bioactive IL-10: Critical role of the cytoplasmic tail in autocrine signaling

The intestinal epithelium is anatomically positioned to serve as the critical interface between the lumen and the mucosal immune system. In addition to MHC class I and II antigens, intestinal epithelia constitutively express the nonclassical MHC molecule CD1d, a transmembrane molecule with a short cytoplasmic tail expressed as a β2-microglobulin-associated 48-kDa glycoprotein and novel β2-microglobulin-independent 37-kDa nonglycosylated protein on intestinal epithelia. At present, it is not known whether extracellular ligands can signal intestinal epithelial CD1d. To define signaling of CD1d cytoplasmic tail, retrovirus-mediated gene transfer was used to generate stable cell lines expressing wild-type CD1d or a chimeric molecule (extracellular CD1d and cytoplasmic CD1a), and surface CD1d was triggered by antibody crosslinking. Although wild-type CD1d was readily activated (tyrosine phosphorylation), no demonstrable signal was evident in cell lines expressing the chimeric molecule. Subsequent studies revealed that anti-CD1d crosslinking specifically induces epithelial IL-10 mRNA and protein and is blocked by the tyrosine kinase inhibitor genistein. Further studies addressing epithelial-derived IL-10 revealed that anti-CD1d crosslinking attenuates IFN-γ signaling and that such attenuation is reversed by addition of functionally inhibitory IL-10 antibodies. These results define signaling through surface CD1d, and, importantly, they demonstrate that this pathway may serve to dampen epithelial proinflammatory signals.

Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation in vitro and in vivo

Small-cell lung carcinoma (SCLC) is an aggressive, rapidly growing and metastasizing, and highly fatal neoplasm. We report that vasoactive intestinal peptide inhibits the proliferation of SCLC cells in culture and dramatically suppresses the growth of SCLC tumor-cell implants in athymic nude mice. In both cases, the inhibition was mediated apparently by a cAMP-dependent mechanism, because the inhibition was enhanced by the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in proportion to increases in intracellular cAMP levels, and the inhibition was abolished by selective inhibition of cAMP-dependent protein kinase. If confirmed in clinical trials, this antiproliferative action of vasoactive intestinal peptide may offer a new and promising means of suppressing SCLC in human subjects, without the toxic side effects of chemotherapeutic agents.

d-myo-Inositol 1,4,5,6-tetrakisphosphate produced in human intestinal epithelial cells in response to Salmonella invasion inhibits phosphoinositide 3-kinase signaling pathways

Several inositol-containing compounds play key roles in receptor-mediated cell signaling events. Here, we describe a function for a specific inositol polyphosphate, d-myo-inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P4], that is produced acutely in response to a receptor-independent process. Thus, infection of intestinal epithelial cells with the enteric pathogen Salmonella, but not with other invasive bacteria, induced a multifold increase in Ins(1,4,5,6)P4 levels. To define a specific function of Ins(1,4,5,6)P4, a membrane-permeant, hydrolyzable ester was used to deliver it to the intracellular compartment, where it antagonized epidermal growth factor (EGF)-induced inhibition of calcium-mediated chloride (Cl−) secretion (CaMCS) in intestinal epithelia. This EGF function is likely mediated through a phosphoinositide 3-kinase (PtdIns3K)-dependent mechanism because the EGF effects are abolished by wortmannin, and three different membrane-permeant esters of the PtdIns3K product phosphatidylinositol 3,4,5-trisphosphate mimicked the EGF effect on CaMCS. We further demonstrate that Ins(1,4,5,6)P4 antagonized EGF signaling downstream of PtdIns3K because Ins(1,4,5,6)P4 interfered with the PtdInsP3 effect on CaMCS without affecting PtdIns3K activity. Thus, elevation of Ins(1,4,5,6)P4 in Salmonella-infected epithelia may promote Cl− flux by antagonizing EGF inhibition mediated through PtdIns3K and PtdInsP3.

Galectin-4 and Small Intestinal Brush Border Enzymes Form Clusters

Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed that galectin-4 is indeed an intestinal brush border protein; we also localized galectin-4 throughout the cell, mainly associated with membraneous structures, including small vesicles, and to the rootlets of microvillar actin filaments. This was confirmed by subcellular fractionation, showing about half the amount of galectin-4 to be in the microvillar fraction, the rest being associated with insoluble intracellular structures. A direct association between the lectin and aminopeptidase N was evidenced by a colocalization along microvilli in double immunogold labeling and by the ability of an antibody to galectin-4 to coimmunoprecipitate aminopeptidase N and sucrase-isomaltase. Furthermore, galectin-4 was released from microvillar, right-side-out vesicles as well as from mucosal explants by a brief wash with 100 mM lactose, confirming its extracellular localization. Galectin-4 is therefore secreted by a nonclassical pathway, and the brush border enzymes represent a novel class of natural ligands for a member of the galectin family. Newly synthesized galectin-4 is rapidly “trapped” by association with intracellular structures prior to its apical secretion, but once externalized, association with brush border enzymes prevents it from being released from the enterocyte into the intestinal lumen.

Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae bile resistance, virulence factor expression, and intestinal colonization

The transmembrane transcriptional activators ToxR and TcpP modulate expression of Vibrio cholerae virulence factors by exerting control over toxT, which encodes the cytoplasmic transcriptional activator of the ctx, tcp, and acf virulence genes. However, ToxR, independently of TcpP and ToxT, activates and represses transcription of the genes encoding two outer-membrane porins, OmpU and OmpT. To determine the role of ToxR-dependent porin regulation in V. cholerae pathogenesis, the ToxR-activated ompU promoter was used to drive ompT transcription in a strain lacking OmpU. Likewise, the ToxR-repressed ompT promoter was used to drive ompU transcription in a strain lacking both ToxR and OmpT. This strategy allowed the generation of a toxR+ strain that expresses OmpT in place of OmpU, and a toxR− strain that expresses OmpU in place of OmpT. Growth rates in the presence of bile salts and other anionic detergents were retarded for the toxR+ V. cholerae expressing OmpT in place of OmpU, but increased in toxR− V. cholerae expressing OmpU in place of OmpT. Additionally, the toxR+ V. cholerae expressing OmpT in place of OmpU expressed less cholera toxin and toxin-coregulated pilus, and this effect was shown to be caused by reduced toxT transcription in this strain. Finally, the toxR+ V. cholerae expressing OmpT in place of OmpU was ≈100-fold reduced in its ability to colonize the infant-mouse intestine. Our results indicate that ToxR-dependent modulation of the outer membrane porins OmpU and OmpT is critical for V. cholerae bile resistance, virulence factor expression, and intestinal colonization.

A resistant genetic background leading to incomplete penetrance of intestinal neoplasia and reduced loss of heterozygosity in ApcMin/+ mice

Previous studies of Min/+ (multiple intestinal neoplasia) mice on a sensitive genetic background, C57BL/6 (B6), showed that adenomas have lost heterozygosity for the germ-line ApcMin mutation in the Apc (adenomatous polyposis coli) gene. We now report that on a strongly resistant genetic background, AKR/J (AKR), Min-induced adenoma multiplicity is reduced by about two orders of magnitude compared with that observed on the B6 background. Somatic treatment with a strong mutagen increases tumor number in AKR Min/+ mice in an age-dependent manner, similar to results previously reported for B6 Min/+ mice. Immunohistochemical analyses indicate that Apc expression is suppressed in all intestinal tumors from both untreated and treated AKR Min/+ mice. However, the mechanism of Apc inactivation in AKR Min/+ mice often differs from that observed for B6 Min/+ mice. Although loss of heterozygosity is observed in some tumors, a significant percentage of tumors showed neither loss of heterozygosity nor Apc truncation mutations. These results extend our understanding of the effects of genetic background on Min-induced tumorigenesis in several ways. First, the AKR strain carries modifiers of Min in addition to Mom1. This combination of AKR modifiers can almost completely suppress spontaneous intestinal tumorigenesis associated with the Min mutation. Second, even on such a highly resistant genetic background, tumor formation continues to involve an absence of Apc function. The means by which Apc function is inactivated is affected by genetic background. Possible scenarios are discussed.

Polyclonal structure of intestinal adenomas in ApcMin/+ mice with concomitant loss of Apc+ from all tumor lineages

When tumors form in intestinal epithelia, it is important to know whether they involve single initiated somatic clones. Advanced carcinomas in humans and mice are known to be monoclonal. However, earlier stages of tumorigenesis may instead involve an interaction between cells that belong to separate somatic clones within the epithelium. The clonality of early tumors has been investigated in mice with an inherited predisposition to intestinal tumors. Analysis of Min (multiple intestinal neoplasia) mice chimeric for a ubiquitously expressed cell lineage marker revealed that normal intestinal crypts are monoclonal, but intestinal adenomas frequently have a polyclonal structure, presenting even when very small as single, focal adenomas composed of at least two somatic lineages. Furthermore, within these polyclonal adenomas, all tumor lineages frequently lose the wild-type Apc allele. These observations can be interpreted by several models for clonal interaction within the epithelium, ranging from passive fusion within regions of high neoplastic potential to a requirement for active clonal cooperation.

T-cell alpha beta + and gamma delta + deficient mice display abnormal but distinct phenotypes toward a natural, widespread infection of the intestinal epithelium.

Vertebrate immune systems contain T cells bearing either alpha beta or gamma delta T-cell antigen receptors (TCRs). alpha beta T cells perform all well-characterized T-cell effector functions, while the biological functions of gamma delta + cells remain unclear. Of particular interest is the role of gamma delta + cells during epithelial infections, since gamma delta + cells are commonly abundant within epithelia. Eimeria spp. are intracellular protozoa that infect epithelia of most vertebrates, causing coccidiosis. This study shows that in response to Eimeria vermiformis, mice lacking alpha beta T cells display defects in protective immunity, while mice lacking gamma delta + cells display exaggerated intestinal damage, apparently due to a failure to regulate the consequences of the alpha beta T cell response. An immuno-downregulatory role during infection, and during autoimmune disease, may be a general one for gamma delta + cells.

Forced expression of the tumor suppressor adenomatosis polyposis coli protein induces disordered cell migration in the intestinal epithelium.

Mutations of the human adenomatosis polyposis coli (APC) gene are associated with the development of familial as well as sporadic intestinal neoplasia. To examine the in vivo function of APC, 129/Sv embryonic stem (ES) cells were transfected with DNA encoding the wild-type human protein under the control of a promoter that is active in all four of the small intestine's principal epithelial lineages during their migration-associated differentiation. ES-APC cells were then introduced into C57BL/6-ROSA26 blastocysts. Analyses of adult B6-ROSA26<-->129/Sv-APC chimeric mice revealed that forced expression of APC results in markedly disordered cell migration. When compared with the effects of forced expression of E-cadherin, the data suggest that APC-catenin and E-cadherin-catenin complexes have opposing effects on intestinal epithelial cell movement/adhesiveness; augmentation of E-cadherin-beta-catenin complexes produces a highly ordered, "adhesive" migration, whereas augmentation of APC-beta-catenin complexes produces a disordered, nonadhesive migratory phenotype. We propose that APC mutations may promote tumorigenesis by increasing the relative activity of cadherin-catenin complexes, resulting in enhanced adhesiveness and functional anchorage of initiated cells within the intestinal crypt. Our studies also indicate that chimeric mice generated from B6-ROSA26 blastocysts and genetically manipulated ES cells should be useful for auditing gene function in the gastrointestinal tract and in other tissues.

Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia.

Giardia lamblia, like most human intestinal parasitic protozoa, sustains fundamental morphological and biochemical changes to survive outside the small intestine of its mammalian host by differentiating into an infective cyst. However, the stimulus that triggers this differentiation remains totally undefined. In this work, we demonstrate the induction of cyst formation in vitro when trophozoites are starved for cholesterol. Expression of cyst wall proteins was detected within encystation-specific secretory vesicles 90 min after the cells were placed in lipoprotein-deficient TYI-S-33 medium. Four cloned lines derived from two independent Giardia isolates were tested, and all formed cysts similarly. Addition of cholesterol, low density or very low density lipoproteins to the lipoprotein-deficient culture medium, inhibited the expression of cyst wall proteins, the generation of encystation-specific vesicles, and cyst wall biogenesis. In contrast, high density lipoproteins, phospholipids, bile salts, or fatty acids had little or no effect. These results indicate that cholesterol starvation is necessary and sufficient for the stimulation of Giardia encystation in vitro and, likely, in the intestine of mammalian hosts.