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PubMed2002

Increased proliferation and apoptosis of colonic epithelial cells in dextran sulfate sodium-induced colitis in rats.

We have evaluated morphologic alterations and epithelial cell apoptosis and proliferation of colonic mucosa in the acute and chronic phases of DSS-induced colitis. Colitis was induced in Sprague-Dawley rats by 7 days of 4% DSS oral administration followed by 7 days of tap water for one, two, and three cycles. Control rats receved tap water only. Morphological changes in colonic mucosa were evaluated and scored by light and scanning electron microscopy. Apoptosis was studied by TUNEL assay and cell proliferation by Ki-67 immunoreaction. The expression of both proapoptotic (Fas, FasL, Bax, p53) and antiapoptotic (Bc12) cellular proteins was determined by immunohistochemistry. Morphologic assessment showed the most severe colonic epithelial lesions and inflammation in the distal colon with a trend to increasing severity from the first to the third DSS cycle. In DSS rats, the epithelial apoptotic index increased 20-fold after the first cycle and 120-fold after the second and third cycles compared with the controls; in the same way, the expression index of proapoptotic proteins (Fas, FasL, Bax, p53) dramatically increased. The proliferative index increased about 40 to 60-fold compared to controls, with no difference among the three DSS cycles. In conclusion, DSS-induced colitis in rats, which has many structural and ultrastructural features similar to those seen in human ulcerative colitis, is a suitable model for studying increased epithelial apoptosis and proliferation. Further studies employing this model will permitt two hypotheses to be tested. (1) Increased apoptosis may lead to a breakdown of the epithelial barrier function and facilitate the mucosal invasion of intraluminal microorganisms and/or antigens. (2) Abnormal and persistent epithelial hyperproliferation could be causally related to the development of colorectal cancers in the setting of chronic colonic inflammation.

Animals↗
PubMed2002

Implication of distinct proteins in cadmium uptake and transport by intestinal cells HT-29.

The mechanisms of intestinal absorption have not been clearly elucidated for cadmium, a toxic metal. In this work, we show the implication of distinct proteins in cadmium transport, and the transport step where these proteins are involved. We first validated the HT-29 model by evaluating nontoxic doses of cadmium (ranging from 1 to 20 micromol/L), and by quantifying metal uptake and transepithelial transport. The time-course of 1 micromol/L cadmium uptake at pH 7.5 showed three steps: a rapid one during the first 4 min, probably due to cadmium binding to the membrane; a slower one, characterized by Km of 1.65+/-0.54 micromol/L and Vmax of 3.9+/-0.3 micromol/min per mg protein; and a third, corresponding to slow accumulation that was not equilibrated even after 48 h of cadmium exposure. Intracellular metallothionein content following 1 or 5 micromol/L cadmium exposure showed a significant increase after 6 h of exposure, and was not equilibrated even after 72 h, allowing cadmium accumulation. After 24 h of exposure, metallothionein content was 5-fold, 14-fold, 26-fold, and 50-fold, respectively, for cells grown in the presence of 1, 5, 10, and 20 micromol/L cadmium, compared to control cells. The second step of uptake, characterized by carrier-mediated transport, was markedly increased at pH 5.5, compared to pH 7.5, and strongly inhibited by the metabolic inhibitor dinitrophenol. Moreover Nramp2 transporter cDNA was present in HT-29 cells. These data suggest the involvement of a proton-coupled transporter, which may be the divalent cation transporter Nramp2 (natural resistance-associated macrophage protein 2). Cadmium uptake was also inhibited by copper, zinc, and para-chloromercuribenzenesulfonate (pCMBS), but not by verapamil or ouabain. Taken together, our results indicate that cadmium could enter HT-29 cell by Nramp2 proton-coupled active transport and by diffusion, and accumulates in the cell as long as it binds to metallothionein. Cadmium toxicity could depend partly on the activity of Nramp2, and partly on metallothionein content.

Biological Transport↗
PubMed2000

Identification of the immunoreactive peptide sequence for AgSK1, an adenocarcinoma-restricted antigen.

SK1, a human immunoglobulin M (IgM) monoclonal antibody was derived from regional nodal lymphocytes of a Dukes B colon carcinoma patient. The antigen recognized by the human monoclonal antibody (HuMab) SK1, termed AgSK1, was shown to be a two-chain glycoprotein with an apparent molecular weight range of 42-46 kDa and preferentially expressed by human adenocarcinomas, particularly human gastrointestinal malignancies. To identify the gene encoding the AgSK1 antigenic epitope, a cDNA expression library constructed in lambda gt22A using mRNA from the colon carcinoma cell line HT29 was screened and one of the isolated clones encoding a 1.5-kb cDNA, which showed strong immunoreactivity with HuMab SK1, was selected for further analysis. This clone consisted of an amino terminal open reading frame of 54 amino acids and the carboxyl terminal 20 amino acids of this protein coding region contained the antigenic epitope recognized by HuMab SK1.

Adenocarcinoma↗
PubMed2005

A colonization factor links Vibrio cholerae environmental survival and human infection.

Many bacteria that cause diseases must be able to survive inside and outside the host. Attachment to and colonization of abiotic or biotic surfaces is a common mechanism by which various microorganisms enhance their ability to survive in diverse environments. Vibrio cholerae is a Gram-negative aquatic bacillus that is often found in the environment attached to the chitinous exoskeletons of zooplankton. It has been suggested that attachment to zooplankton enhances environmental survival of Vibrio spp., probably by providing both an abundant source of carbon and nitrogen and protection from numerous environmental challenges. On ingestion by humans, some serogroups of V. cholerae cause the diarrhoeal disease cholera. The pathophysiology of cholera is a result of the effects of cholera toxin on intestinal epithelial cells. For sufficient quantities of cholera toxin to reach the intestinal epithelium and to produce clinical symptoms, colonization of the small bowel must occur. Because most V. cholerae do not colonize humans, but all probably require strategies for survival in the environment, we considered that colonization factors selected for in the environment may be the same as those required for intestinal colonization of humans. In support of this hypothesis, here we have identified a single protein required for efficient intestinal colonization that mediates attachment to both zooplankton and human epithelial cells by binding to a sugar present on both surfaces.

Acetylglucosamine↗
PubMed2004

Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation.

Wnt signaling defines the colonic epithelial progenitor cell phenotype, and mutations in the gene adenomatous polyposis coli (APC) that activate the Wnt pathway cause the familial adenomatous polyposis coli (FAP) syndrome and most sporadic colon cancers. The mechanisms that regulate the transition of epithelial precursor cells into their differentiated derivatives are poorly characterized. We report that Indian hedgehog (Ihh) is expressed by mature colonocytes and regulates their differentiation in vitro and in vivo. Hedgehog (Hh) signaling restricts the expression of Wnt targets to the base of the colonic crypt in vivo, and transfection of Ihh into colon cancer cells leads to a downregulation of both components of the nuclear TCF4-beta-catenin complex and abrogates endogenous Wnt signaling in vitro. In turn, expression of Ihh is downregulated in polyps of individuals with FAP and expression of doxycycline-inducible dominant negative TCF4 (dnTCF4) restores Ihh expression in APC mutant DLD-1 colon cancer cells. These data identify a new Wnt-Hh axis in colonic epithelial renewal.

Adenomatous Polyposis Coli↗
PubMed2003

Hypermethylation of the gene promoter and enhancer region can regulate Fas expression and sensitivity in colon carcinoma.

Expression of the cell surface receptor Fas is frequently lost or decreased during tumor progression in human colon carcinomas. The methylation status of a 583 bp CpG-rich region within the Fas promoter (-575 to +8) containing 28 CpG sites was determined in human colon carcinoma cell lines. In Caco(2) (no Fas expression), 82-93% of CpG sites were methylated, whereas none were methylated in GC(3)/c1 (high Fas expression). In RKO (intermediate level of Fas), a single CpG site, located at -548, was 100% methylated. The inhibitor of methylation, 5-aza-2'-deoxycytidine (5-azadC), upregulated Fas expression in four of eight cell lines, and sensitized RKO cells to recombinant FasL-induced apoptosis. The p53-binding region in the first intron of the Fas gene was partially methylated in Caco(2), and 5-azadC potentiated Ad-wtp53-induced upregulation of Fas expression. Methylation-specific PCR of the first intron detected partial methylation in four out of 10 colon carcinoma tumor samples in vivo. The data suggest that DNA hypermethylation is one mechanism that contributes to the downregulation of Fas expression and subsequent loss of sensitivity to Fas-induced apoptosis in colon carcinoma cells.

Apoptosis↗
PubMed2006

The antitumor activity of TRAIL and IL-24 with replicating oncolytic adenovirus in colorectal cancer.

Melanoma differentiation associated gene-7 (Mda-7)/IL-24 was previously cloned into ZD55 (an adenovirus with E1B55 deleted) to form ZD55-IL-24, which had much better antitumor effect than Ad-IL-24. According to its good antitumor properties, ZD55-IL-24 has been used in preclinical studies. But ZD55-IL-24 alone still could not completely eradicate established tumors in all nude mice. It was reported that IL-24 could induce and enhance the activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (a member of tumor necrosis factor (TNF) superfamily). Accordingly, the combined use of ZD55-IL-24 and ZD55-TRAIL was carried out in this study. Treatment with both ZD55-IL-24 and ZD55-TRAIL could induce more significant apoptosis in cancer cells in vitro compared with ZD55-IL-24 or ZD55-TRAIL alone. The combination of the two replicative adenoviruses had better antitumor activity in vivo than that of single oncolytic adenovirus and led to complete eradication of xenograft tumors in all treated mice. Upregulation of TRAIL was observed in tumor cells infected with ZD55-IL-24 and studies of the apoptotic cascade regulators indicate that ZD55-IL-24 could further enhance the activation of apoptosis through the TNF family of death receptors. We demonstrated for the first time the potential therapeutic effect of combined ZD55-IL-24 with ZD55-TRAIL for the targeted therapy of cancer.

Adenoviridae↗
PubMed1999

Mutations in the promoter reveal a cause for the reduced expression of the human manganese superoxide dismutase gene in cancer cells.

Manganese superoxide dismutase (MnSOD) has been shown to play an important role in preventing the development of cancer. MnSOD activity is reduced in many transformed cells and tumor tissues. We previously showed that the reduced level of MnSOD activity in cancer cells was not due to a defect in the primary structure of MnSOD protein, but rather was due to defects in gene expression. To elucidate the cause for the reduced expression of human MnSOD in cancer, we investigated the nucleotide sequence in the regulatory region of the MnSOD gene in a normal human cell line and various human tumor cell lines. A DNA fragment spanning 3.4 kb 5' flanking region of the MnSOD gene isolated from a normal human genomic DNA library was used to determine the DNA sequence of MnSOD promoter. PCR primers were used for amplification of the 3.4 kb 5' flanking region of the human MnSOD gene in cancer cells. Sequence analysis identified three heterozygous mutations in the proximal region of the promoter in five human tumor cell lines. These mutations, clustered around the GC-rich region of the human MnSOD promoter, change the binding pattern of AP-2 and lead to a reduction in transcription activity using a luciferase reporter assay system. These results suggest that the reduced level of MnSOD expression in some tumor cells is, at least in part, due to a defect in the DNA sequence of the promoter region.

Base Sequence↗
PubMed2005

Commutators of PAR-1 signaling in cancer cell invasion reveal an essential role of the Rho-Rho kinase axis and tumor microenvironment.

We recently reported that proteinase-activated receptors type I (PAR-1) are coupled to both negative and positive invasion pathways in colonic and kidney cancer cells cultured on collagen type I gels. Here, we found that treatments with the cell-permeant analog 8-Br-cGMP and the soluble guanylate cyclase activator BAY41-2272, and Rho kinase (ROK) inhibition by Y27632 or a dominant negative form of ROK lead to PAR-1-mediated invasion through differential Rac1 and Cdc42 signaling. Hypoxia or the counteradhesive matricellular protein SPARC/BM-40 (SPARC: secreted protein acidic rich in cysteine) overexpressed during cancer progression also commutated PAR-1 to cellular invasion through the cGMP/protein kinase G (PKG) cascade, RhoA inactivation, and Rac1-dependent or -independent signaling. Cultured primary cancer cells isolated from peritoneal and pleural effusions from patients with colon cancer or other malignant tumors harbored PAR-1, as shown by RT-PCR and FACS analyses. These malignant effusions also contained high levels of activated thrombin and fibrin, and induced a proinvasive response in HCT8/S11 human colorectal cancer cells. Our data underline the essential role of the tumor microenvironment and of several commutators targeting cGMP/PKG signaling and the RhoA-ROK axis in the control of PAR-1 proinvasive activity and metastatic potential of cancer cells in distant organs and peritoneal or pleural cavities. We also add new insights into the mechanisms linking the coagulation mediators thrombin and PAR-1 in the context of blood coagulation disorders and venous thrombosis often observed in cancer patients, as described in 1865 by Armand Trousseau.

Aged↗
PubMed2006

Endogenous production of leukotriene D4 mediates autocrine survival and proliferation via CysLT1 receptor signalling in intestinal epithelial cells.

The cysteinyl leukotriene1 (CysLT1) receptor (CysLT1R) enhances survival and proliferation of intestinal cells via distinct pathways. Here, we have demonstrated that there is significant endogenous production of CysLTs from both non-tumour- and tumour-derived intestinal epithelial cells. Treatment of two non-tumour cell lines, Int 407 and IEC-6, with CysLT1R antagonists led to shrinkage and detachment of cells, confirmed as apoptotic cell death, and a dose-dependent reduction in proliferation. However, in the tumour intestinal cell lines Caco-2, SW480, HCT-116 and HT-29, treatment with CysLT1R antagonists significantly reduced proliferation, but had no effect on apoptosis. A unique characteristic of intestinal cancer cells is the presence of nuclear CysLT1Rs, which are inaccessible to receptor antagonists. In these cells, inhibition of the endogenous production of CysLTs indirectly, by 5-lipoxygenase inhibition, impaired CysLT1R signalling throughout the cell, and resulted in apoptosis of the tumour cells. These data reveal the existence of constitutive CysLT1R signalling that mediates both survival and proliferation in intestinal cells. Importantly, we propose that tumour-derived intestinal cells are resistant to CysLT1R antagonist-induced apoptosis, a phenomena that could be explained by nuclear CysLT1R signalling.

Apoptosis↗
PubMed2006

Upregulation of BAK by butyrate in the colon is associated with increased Sp3 binding.

Butyrate is a key bioactive product of dietary fibre fermentation thought to play a key role in cancer prevention. One contributory mechanism in this role is the regulation of apoptosis by butyrate. As butyrate shows low levels of toxicity, the mechanisms by which it triggers or regulates apoptosis are of great interest. We and others have shown that the proapoptotic protein BAK is upregulated by butyrate. We show here that this observation is conserved across multiple cell lines, that it occurs in all cells in a population and is at the transcriptional level. We have used a promoter-reporter construct to identify the regulatory regions of the BAK promoter and found that much of the transcriptional activity occurs via a single Sp1/Sp3 binding site. We have shown that both Sp1 and Sp3 bind, but upon butyrate treatment Sp1 binding decreases in favour of Sp3 binding. We speculate that this may be an acetylation-mediated event.

Apoptosis↗
PubMed2006

Frequent requirement of hedgehog signaling in non-small cell lung carcinoma.

Although it had previously been suggested that the hedgehog (HH) pathway might be activated in some lung tumors, the dependence of non-small cell lung carcinomas (NSCLC) for HH activity had not been comprehensively studied. During a screen of a panel of 60 human tumor cell lines with an HH antagonist, we observed that the proliferation of a subset of NSCLC cell lines was inhibited. These NSCLC cell lines express HH, as well as key HH target genes, consistent with them being activated through an autocrine mechanism. Interestingly, we also identified a number of NSCLC cell lines that express high levels of the downstream transcription factor GLI1 and harbor enhanced levels of HH activity, but appear insensitive to known HH antagonists. We hypothesized that the high levels of GLI1 in these cells would function downstream of the HH antagonist target, allowing them to bypass the antagonist-mediated block in proliferation. Consistent with this hypothesis, when the levels of GLI1 are knocked down in such cells, they become sensitive to these inhibitors. We go on to show that a large percentage of primary NSCLC samples express GLI1, consistent with constitutive activation of the HH pathway in these samples. Taken together, these results establish the involvement of the HH signaling pathway in a subset of NSCLCs.

Carcinoma, Non-Small-Cell Lung↗
PubMed1998

Sequence of the cDNA of a human dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA.

Human liver contains three isoforms (DD1, DD2 and DD4) of dihydrodiol dehydrogenase with 20alpha- or 3alpha-hydroxysteroid dehydrogenase activity; the dehydrogenases belong to the aldo-oxo reductase (AKR) superfamily. cDNA species encoding DD1 and DD4 have been identified. However, four cDNA species with more than 99% sequence identity have been cloned and are compatible with a partial amino acid sequence of DD2. In this study we have isolated a cDNA clone encoding DD2, which was confirmed by comparison of the properties of the recombinant and hepatic enzymes. This cDNA showed differences of one, two, four and five nucleotides from the previously reported four cDNA species for a dehydrogenase of human colon carcinoma HT29 cells, human prostatic 3alpha-hydroxysteroid dehydrogenase, a human liver 3alpha-hydroxysteroid dehydrogenase-like protein and chlordecone reductase-like protein respectively. Expression of mRNA species for the five similar cDNA species in 20 liver samples and 10 other different tissue samples was examined by reverse transcriptase-mediated PCR with specific primers followed by diagnostic restriction with endonucleases. All the tissues expressed only one mRNA species corresponding to the newly identified cDNA for DD2: mRNA transcripts corresponding to the other cDNA species were not detected. We suggest that the new cDNA is derived from the principal gene for DD2, which has been named AKR1C2 by a new nomenclature for the AKR superfamily. It is possible that some of the other cDNA species previously reported are rare allelic variants of this gene.

Base Sequence↗
PubMed1996

E-selectin: sialyl Lewis, a dependent adhesion of colon cancer cells, is inhibited differently by antibodies against E-selectin ligands.

Recent studies have demonstrated that selectins, a new family of cell-adhesion molecules with similar domain structures, mediate the adhesion of peripheral blood cells to interleukin-1 (IL-1)-activated endothelium. In the present study the authors evaluated the role of E-selectin-Sialyl Lewis x (SLe(x))/ Sialyl Lewis a (SLe(a)) interaction in mediating in vitro adhesion of two colon cancer cell lines, HT-29 and COLO 201, to human umbilical cord endothelial cells (HUVEC). Colon cancer cell lines had a strong expression of blood group-related carbohydrate epitopes as evaluated by fluorescence-activated cell sorter (FACS) analysis. It was established that adhesion of HT-29 and COLO 201 cells to IL-1 stimulated HUVEC was calcium dependent and could be inhibited by a monoclonal antibody directed against E-selectin. Prior incubation of cells with two different antibodies directed against SLe(x) and antibodies directed against related Lewis epitopes, Le(x) and Le(a), had no significant effect on adhesion. Three antibodies directed against SLe(a) differed in their capacity to inhibit the adhesion of HT-29 and COLO 201 cells to HUVEC. Only one antibody directed against the SLe(a) structure was effective in inhibiting adhesion of both COLO 201 and HT-29 cells. The difference could not be attributed to titre, the type or number of glycoproteins, or to a difference in the amount of SLe(a) present on individual proteins, suggesting that presence and right presentation of SLe(a) epitope might be important for adhesion of colon cancer cells. Finally, in the in vitro system used, adhesion of HT-29 and COLO 201 cells to activated HUVEC is mediated predominantly by E-selectin/SLe(a) interaction. SLe(x) and related epitopes, Le(x) and Le(a), seem to have limited relevance for colon cancer cell recognition of E-selectin.

Antibodies, Monoclonal↗
PubMed2004

Enhanced Escherichia coli adherence and invasion in Crohn's disease and colon cancer.

BACKGROUND & AIMS: Altered mucosal glycosylation in inflammatory bowel disease and colon cancer could affect mucosal bacterial adherence. This study aimed to quantify and characterize mucosa-associated and intramucosal bacteria, particularly Escherichia coli, in these conditions. METHODS: Mucosa-associated bacteria were isolated, after dithiothreitol mucolysis, from biopsy samples obtained at colonoscopy (Crohn's disease, n = 14 patients; ulcerative colitis, n = 21; noninflamed controls, n = 24) and at surgical resection (colon cancer, n = 21). Intramucosal bacteria were grown after gentamicin treatment followed by hypotonic lysis. RESULTS: Mucosa-associated and intramucosal bacteria were cultured more commonly in Crohn's disease (79%, P = 0.03; and 71%, P < 0.01, respectively), but not ulcerative colitis (38% and 48%), than in noninflamed controls (42% and 29%) and were commonly cultured from colon cancers (71% and 57%). Mucosa-associated E. coli, which accounted for 53% of isolates, were more common in Crohn's disease (6/14; 43%) than in noninflamed controls (4/24, 17%), as also were intramucosal E. coli: Crohn's disease, 29%; controls, 9%. E. coli expressed hemagglutinins in 39% of Crohn's cases and 38% of cancers but only 4% of controls, and this correlated (P = 0.01) with adherence to the I407 and HT29 cell lines. Invasion was cell-line dependent. E. coli, including nonadherent isolates, induced interleukin-8 release from the cell lines. E. coli adhesins showed no blood group specificity, excepting 1 cancer isolate (HM44) with specificity for the Thomsen-Friedenreich antigen, but they could be blocked by soluble plantain fiber. CONCLUSIONS: These studies support a central role for mucosally adherent bacteria in the pathogenesis of Crohn's disease and colon cancer. Soluble plant fibers that inhibit their adherence have therapeutic potential.

Bacterial Adhesion↗
PubMed2000

Sensitivity to CPT-11 of xenografted human colorectal cancers as a function of microsatellite instability and p53 status.

Biological parameters influencing the response of human colorectal cancers (CRCs) to CPT-11, a topoisomerase 1 (top1) inhibitor, were investigated using a panel of nine CRCs xenografted into nude mice. CRC xenografts differed in their p53 status (wt or muf) and in their microsatellite instability phenotype (MSI+ when altered). Five CRC xenografts were established from clinical samples. All five had a functional p53, two were MSI+ and three were MSI-. Tumour-bearing nude mice were treated intraperitonealy (i.p.) with CPT-11. At 10 mg kg(-1) of CPT-11, four injections at 4-day intervals, four of the five xenografts responded to CPT-11 (growth delay of up to 10 days); the non-responder tumour was MSI-. At 40 mg kg(-1) of CPT-11, six injections at 4-day intervals, the five CRCs displayed variable but marked responses with complete regressions. In order to assess the role of p53 status in CPT-11 response, four CRC lines were used. HT29 cell line was MSI-/Ala273-mutp53, its subclone HT29A3 being transfected by wtp53. LoVo cell line was MSI+/wtp53, its subclone X17LoVo dominantly expressed Ala273-mutp53 after transfection. LoVo tumours (MSI+/mutp53) were more sensitive than X17LoVo (MSI+/mutp53. HT 29 tumours (MSI-Imutp53), were refractory to CPT-11 while HT29A3 tumours (MSI-/wtp53) were sensitive, showing that wtp53 improves the drug-response in these MSI- tumours. Levels of mRNA expression of top1, fasR, TP53 and mdr1 were semi-quantified by reverse transcription polymerase chain reaction. None of these parameters correlated with CPT-11 response. Taken together, these observations indicate that MSI and p53 alterations could be associated with different CPT-11 sensitivities; MSI phenotype moderately influences the CPT-11 sensitivity, MSI+ being more sensitive than MSI(-)CRC freshly obtained from patients, mutp53 status being associated with a poor response to CPT-11.

Animals↗
PubMed1996

Gadolinium(III) texaphyrin: a tumor selective radiation sensitizer that is detectable by MRI.

Gadolinium(III) texaphyrin (Gd-tex2+) is representative of a new class of radiation sensitizers detectable by magnetic resonance imaging (MRI). This porphyrin-like complex has a high electron affinity [E1/2 (red.) approximately = -0.08 V versus normal hydrogen electrode] and forms a long-lived pi-radical cation upon exposure to hydrated electrons, reducing ketyl radicals, or superoxide ions. Consistent with these chemical findings, Gd-tex2+ was found to be an efficient radiation sensitizer in studies carried out with HT29 cells in in vitro as well as in in vivo single and multifraction irradiation studies with a murine mammary carcinoma model. Selective localization of Gd-tex2+ in tumors was confirmed by MRI scanning.

Animals↗
PubMed1999

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 beta(2)-microglobulin-associated 48-kDa glycoprotein and novel beta(2)-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-gamma 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.

Antigens, CD1↗