Subversion of human intestinal mucosa innate immunity by a Crohn's disease-associated E. coli.

Adherent-invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82 on two innate immunity platforms, i.e., the inflammasome through evaluation of caspase-1 status, and NFκB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and macrophages, without altering the mucosa overall architecture. Although 4-h infection with a Salmonella strain caused crypt disorganization, caspase-1 activation, and mature IL-18 production, LF82 bacteria were unable to activate caspase-1 and induce IL-18 production. In parallel, LF82 bacteria activated NFκB signaling in epithelial cells through IκBα phosphorylation, NFκBp65 nuclear translocation, and TNFα secretion. In addition, NFκB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole-mucosa level the mechanisms of the LF82-induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

Altered Calreticulin expression in human colon cancer: maintenance of Calreticulin expression is associated with mucinous differentiation.

Calreticulin is an endoplasmic reticulum luminal calcium-binding chaperone involved in various cellular functions and is a ligand for the scavenger receptor CD91. Recent studies, based on proteomic approaches on whole tissue samples containing both neoplastic and non-neoplastic cells, have shown alterations of Calreticulin expression in colon carcinomas, albeit with divergent results. The aims of this study were: 1) to assess the expression of Calreticulin and its receptor CD91 in 58 human colon adenocarcinomas, compared with paired normal mucosa, using a semi-quantitative immunohistochemical analysis, and 2) to examine associations between the tumour phenotypic features, and Calreticulin and/or CD91 expressions. Calreticulin expression was down-regulated in 51.7% human colon adenocarcinomas. Accordingly, quantitative immunoblot analysis showed that Calreticulin expression was significantly lower in human colonic cancer cell lines than in preparations of isolated human normal colonic epithelial cells. CD91 was co-expressed with Calreticulin in both normal colonic epithelial cells and pericryptic myofibroblasts. Calreticulin and CD91, that characterize the 'amateur phagocyte' function of epithelial cells, were both down-regulated in 48% of adenocarcinomas. Finally, Calreticulin expression was significantly associated with the mucinous differentiation of the tumour. Collectively, these results show that Calreticulin is likely to play a pivotal role in the differentiation of human colonic adenocarcinomas.

Phosphohistone H3 labelling for histoprognostic grading of breast adenocarcinomas and computer-assisted determination of mitotic index.

BACKGROUND: Microscopic evaluation of mitotic figures is a routine procedure in the assessment of the histoprognostic grade of tumours. Nevertheless, their count may be fraught with difficulties. As histone H3 phosphorylation at serine 10 is closely linked to chromosomal condensation, a new monoclonal antibody directed to phosphorylated histone H3 (PPH3) was recently proposed to detect mitotic cells. AIM: To test the reliability of this antibody in detecting and counting mitotic figures in sections of breast adenocarcinomas, because of the importance of mitotic count in histoprognostic grading. METHODS: The pattern of PPH3 staining in formalin-fixed paraffin wax-embedded tissues, including normal tissues and a series of 39 breast adenocarcinomas, was examined. A new computer-assisted method was also developed for determining the mitotic index. RESULTS AND CONCLUSIONS: In all tissues tested, PPH3-labelled mitotic figures were easily detected, allowing a rapid identification of the area of highest mitotic activity. In breast carcinomas, a strong correlation was observed between PPH3-stained and haematoxylin and eosin-stained mitotic counts (r = 0.86, p<0.0001). Counting of prophase nuclei that coexpress cyclin B1, a marker of the G2/M phase, was possible by PPH3 staining; its accuracy led us to reconsider the tumour grade in three cases. Finally, an automatic computer-assisted method was designed for assessing mitotic index with confocal microscopy and image-analysis software.

Antibiotic therapy reduces nitrosative stress and programmed cell death in the rabbit foetal lung.

The correlation of clinical and epidemiological data suggests that intrauterine infection/inflammation can promote foetal lung injury. The aim of this study was: 1) to characterise the early inflammatory response elicited in infected foetal lungs, in terms of nitric oxide-derived oxidative stress and programmed cell death; and 2) to investigate the effects of antibiotic therapy on these parameters. A previously described rabbit experimental model of materno-foetal infection was used. Animals were divided into three groups: controls; Escherichia coli infected (12 h); and E. Coli infected (12 h) and treated (24 h gentamicin+ceftriaxone). Foetal lungs were examined in terms of histology, nitric oxide synthase (NOS) activity, immunohistochemical detection of 3-nitrotyrosine, and detection of apoptotic cells by the TUNEL assay and Hoechst staining. In the infected group, a moderate inflammatory response was observed, associated with a significant increase in inducible NOS activity, the formation of 3-nitrotyrosine residues in epithelial and immune cells, the down-regulation of constitutive NOS activity and clusters of apoptotic cells, as compared with the control group. Early antibiotic therapy, initiated at 12 h post-inoculation, elicited a significant decrease in the infection-induced nitrosative stress. Levels of 3-nitrotyrosine and of apoptotic cells were decreased in the infected-and-treated group compared with the infected group, mainly by the re-expression of constitutive NOS and of the basal level of inducible NOS. Altogether, these findings indicate that early antibiotic therapy can curb the inflammatory reaction and help avert antenatal lung injury, which is known to be involved in the onset of bronchopulmonary dysplasia.

Loss of NOS1 expression in high-grade renal cell carcinoma associated with a shift of NO signalling.

In normal human kidney, NOS1 and soluble guanylate cyclase (sGC) are expressed in tubular epithelial cells, suggesting a physiological autocrine NO signalling pathway. Therefore, we investigated both NOS1 and sGC expressions in benign and malignant renal tumours. In addition, we examined the pattern of protein tyrosine nitration in normal and tumour tissue. NOS1 expression and activity were found to be downregulated, correlating with the tumour grade, as shown by immunohistochemistry, quantitative RT-PCR analysis, and histochemical detection of the NADPH-diaphorase activity of nitric oxide synthases (NOS). These results show that the autocrine NO signalling pathway is maintained in benign tumours and lost in malignant tumours. In contrast, sGC expression was maintained in renal tumours whatever the tumour type, a finding showing that tumour cells remain sensitive to the bioregulatory role of exogeneous NO(*). Finally, the staining pattern of protein tyrosine nitration, assessed by immunohistochemistry, parallelled that of NOS1 expression in normal renal parenchyma and benign tumours, supporting the concept that protein nitration was accounted for by NOS1 activity. In contrast, in malignant tumours, protein tyrosine nitration was accounted for by the production of reactive nitrogen oxide species by the inflammatory infiltrate. Altogether, these findings argue for a pattern of NO signalling similar in normal kidney and benign renal tumours, whereas it is completely different in malignant renal tumours.

Human submucosal neurones regulate intestinal epithelial cell proliferation: evidence from a novel co-culture model.

The role of the human enteric nervous system (ENS) in the control of the intestinal epithelium organization and proliferation is unknown. To address this issue, we developed a novel co-culture model, consisting of human submucosa containing the submucosal plexus and a human colonic epithelial monolayer. After 3 days in basal conditions (i.e. in absence of neuronal activation) epithelium disorganization and proliferation occurred. In contrast, electrical activation of submucosal neurones maintained monolayer organization and decreased cell proliferation. These effects were blocked by tetrodotoxin and a vasoactive intestinal peptide (VIP) receptor antagonist, and reproduced by VIP. In conclusion, our study suggests that the human ENS is involved in the control of epithelial cell proliferation.

The in vitro manipulation of carbohydrate metabolism: a new strategy for deciphering the cellular defence mechanisms against nitric oxide attack.

This study was aimed at examining the effects of manipulating the carbohydrate source of the culture medium on the cellular sensitivity of epithelial cells to an oxidative attack. Our rationale was that substituting galactose for glucose in culture media would remove the protection afforded by glucose utilization in two major metabolic pathways, i.e. anaerobic glycolysis and/or the pentose phosphate pathway (PPP), which builds up cellular reducing power. Indeed, we show that the polarized human colonic epithelial cell line HT29-Cl.16E was sensitive to the deleterious effects of the NO donor PAPANONOate [3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine] only in galactose-containing medium. In such medium NO attack led to cytotoxic and apoptotic cell death, associated with formation of derivatives of NO auto-oxidation (collectively termed NOx) and peroxynitrite, leading to intracellular GSH depletion and nitrotyrosine formation. The addition of 2-deoxyglucose, a non-glycolytic substrate, to galactose-fed cells protected HT29-Cl. 16E cells from NO attack and maintained control GSH levels through its metabolic utilization in the PPP, as shown by (14)CO(2) production from 2-deoxy[1-(14)C]glucose. Therefore, increasing the availability of reducing equivalents without interfering with energy metabolism is able to prevent NO-induced cell injury. Finally, this background provides the conceptual framework for establishing nutritional manipulation of cellular metabolic pathways that could provide new means for (i) deciphering the mechanisms of cell injury by reactive nitrogen species and reactive oxygen species at the whole-cell level and (ii) establishing the hierarchy of intracellular defence mechanisms against these attacks.

Interleukin 1 and interleukin 1beta converting enzyme (caspase 1) expression in the human colonic epithelial barrier. Caspase 1 downregulation in colon cancer.

BACKGROUND: Interleukin (IL) 1beta converting enzyme (now known as caspase 1) is able to process pro-IL-1beta into its active form and is involved in proapoptotic signalling. AIMS: To characterise IL-1 and caspase 1 expression in human colonic epithelial cells. METHODS: Intracellular IL-1 content (IL-1alpha and IL-1beta) was measured by ELISA in freshly isolated human normal colonocytes. Caspase 1 expression was determined both at the mRNA level using in situ hybridisation and reverse transcription polymerase chain reaction, and at the protein level by immunoblotting experiments using antibodies specific for the proform of caspase 1 and for its cleavage forms. RESULTS: Low amounts of IL-1beta were found in nearly all preparations (92%), and IL-1alpha was detected in only 45% of human colonocyte preparations. The normal colonic epithelium strongly expressed caspase 1, both at the mRNA level and at the protein level in its latent form. In contrast, caspase 1 was not expressed in colon cancer (primary colonic adenocarcinomas and cancer cell lines). CONCLUSIONS: The demonstration that the human colonic epithelial barrier is able to express caspase 1 and its substrate IL-1beta reinforces the concept that, under certain conditions, the epithelium could trigger an inflammatory reaction. In addition, the finding that caspase 1 was downregulated in colonic adenocarcinomas supports the concept that disrupted apoptosis pathways may be involved in tumour formation and/or may confer resistance to treatment.

Purinergic and cholinergic agonists induce exocytosis from the same granule pool in HT29-Cl.16E monolayers.

Several secretagogues induce mucin secretion in epithelial monolayers, as determined by measuring released granule contents. To assess whether different agonists act on the same granule pool, capacitance changes in intact monolayers of the goblet cell line HT29-Cl.16E were measured by a novel impedance method. Apical ATP (purinergic agonist) and basolateral carbachol (cholinergic agonist) induce rapid exocytosis with maximal capacitance changes within 3 min. The maximal levels of exocytosis that can be induced by optimal concentrations of either agonist are the same and produce a 30-40% increase in total monolayer capacitance. When ATP and carbachol are applied simultaneously, the magnitude of exocytosis is unchanged from the single-secretagogue level. The recovery of capacitance to baseline (endocytosis) is significantly faster after ATP stimulation than after carbachol stimulation. When ATP and carbachol are applied sequentially at doses that give maximal exocytosis, the magnitude of the capacitance increase produced by the second secretagogue is less than or equal to that of the capacitance decrease during the recovery period. Together, these data suggest that purinergic and cholinergic agonists act on the same granule pool.

Listeria monocytogenes stimulates mucus exocytosis in cultured human polarized mucosecreting intestinal cells through action of listeriolysin O.

When the intracellular pathogen Listeria monocytogenes infects cultured human mucosecreting polarized HT29-MTX cells apically, it induces the stimulation of mucus exocytosis without cell entry. Using a set of isogenic mutants and purified listeriolysin O (LLO), we identified the L. monocytogenes thiol-activated exotoxin LLO as the agonist of mucus secretion. We demonstrated that the LLO-induced mucus exocytosis did not result from the LLO membrane-damaging activity. We found that LLO-induced mucus exocytosis is an event requiring the binding of LLO to a brush border-associated receptor and membrane oligomerization of the exotoxin. By a pharmacological approach, we demonstrated that no regulatory system or intracellular transducing signal known to be involved in control of mucin exocytosis was activated by LLO. Based on the present data, the stimulatory action of LLO on mucin exocytosis could be accounted for either by an unknown signaling system which remains to be determined or by direct action of LLO with the membrane vesicle components involved in the intracellular vesicular transport of mucins.

Control of growth and differentiation of normal human epithelial cells through the manipulation of reactive nitrogen species.

In this work, we addressed the issue of whether exogenous NO and ONOO- (peroxynitrite) are able to alter growth, viability and/or differentiation of normal epithelial cells using cultured normal human keratinocytes as a model. 3-Morpholino-sydnonimine (SIN-1), a donor of both NO and O2(-)., leading to the production of ONOO-, dose-dependently inhibited growth of human keratinocytes without loss of viability. This inhibitory effect was lowered when SIN-1 was transformed into a pure NO donor by scavenging O2(-). with superoxide dismutase/catalase. Finally, scavenging NO release from SIN-1 with carboxy-1H-imidazol-1-yloxy,2-(4-carboxyp henyl)-4,5-dihydro-4,4,5,5 -tetramethyl-3-oxide (PTIO) resulted in a loss of the inhibitory effect of SIN-1. Together these findings suggest that both ONOO- and NO exert a growth inhibitory effect on human keratinocytes without cytotoxicity. Further support for this conclusion came from the treatment of human keratinocytes with the NO. donor propanamine 3-(2-hydroxy-2-nitroso-1-propyl hydrazino) or with authentic peroxynitrite. Moreover, only SIN-1 or peroxynitrite, and not NO, was able to trigger the expression of markers of terminal differentiation in human keratinocytes. From a physiological perspective, the ability of peroxynitrite, a known genotoxic and potentially carcinogenic agent, to direct proliferating keratinocytes towards terminal differentiation may be crucial to protect the genomic stability of this barrier epithelium.

Early functional effects of Clostridium difficile toxin A on human colonocytes.

BACKGROUND & AIMS: Previous in vitro studies have shown that Clostridium difficile toxin A is able to directly affect the intestinal epithelial barrier function. The aim of this study was to examine the early effects of toxin A on mucin exocytosis and determine whether this toxin can induce the production of the chemokine interleukin 8 (IL-8) from human colonic epithelial cells. METHODS: Two model systems were used: the HT29-CI.16E colonic goblet cell line and primary cultures of human normal colonocytes. RESULTS: Toxin A exerted a rapid and dose-related inhibition of stimulated mucin exocytosis without altering baseline (constitutive) mucin exocytosis from HT29-CI.16E cells. Toxin A was also able to induce the secretion of IL-8 from both HT29-CI.16E cells and primary cultures of human normal colonocytes, as early as 2-3 hours of incubation. CONCLUSIONS: The results show that while toxin A is able to down-regulate stimulated mucin exocytosis, it is able to up-regulate the secretion of an important chemoattractant chemokine, IL-8. These modifications illustrate the ability of colonocytes to recruit inflammatory and immune cells that will eventually bring about major mucosal damage.

NO-dependent and NO-independent IL-1 production by a human colonic epithelial cell line under inflammatory stress.

1. The present study was designed to investigate, in an in vitro model of the human intestinal barrier, the ability of epithelial cells to produce interleukin-1 (IL-1), the cellular mechanisms involved in IL-1 release, and the intracellular signalling pathways involved in IL-1 up-regulation during inflammatory stress. 2. This study was based on the human colonic epithelial cell line HT29-Cl.16E, maintained as polarized monolayers on filters mounted in culture chambers and treated with various proinflammatory cytokines (interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha), IL-1 beta) alone or in combination. 3. IL-1 production, restricted to IL-1 alpha, was induced by the combination of IFN gamma/TNF alpha. When IL-1 beta was added to IFN gamma/TNF alpha, it led to an additional production of IL-1 alpha. IL-1 alpha release was associated with cell damage, as shown by the correlation between lactate dehydrogenase (LDH) release and extracellular IL-1 production, and was not accounted for by a secretory mechanism. 4. Both IFN gamma/TNF alpha and IFN gamma/TNF alpha/IL-1 beta induced inducible nitric oxide synthase (iNOS) expression as shown by quantitation of NO2-/NO3- by use of the Griess reagent, quantitation of cells scoring positive with an anti-iNOS antibody and detection of mRNAs coding for iNOS by RT-PCR. The use of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS, led to the demonstration of two distinct signalling pathways in IL-1 production by HT29-Cl.16E cells, one dependent on NO (L-NMMA-sensitive) under treatment with IFN gamma/TNF alpha/IL-1 beta, and the other independent of NO (L-NMMA-insensitive) under treatment with IFN gamma/TNF alpha. 5. Moreover, we examined whether a redox-based mechanism could be responsible for the apparent discrepancy between NO production and NO implication in IL-1 production under IFN gamma/TNF alpha and IFN gamma/TNF alpha/IL-1 beta treatments. Experiments with cysteine, which acts as a powerful reductant, suggest that the nitrosonium character of NO is involved in the NO-dependent pathway in IL-1 production.

Stimulation of mucin exocytosis from human epithelial cells by nitric oxide: evidence for a cGMP-dependent and a cGMP-independent pathway.

The aim of this work was to investigate the role of nitric oxide (NO) on the macromolecular exocytotic function of human epithelial cells. We tested the effects of two NO-generating drugs, i.e. 1-hexanamine 6-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl (MAHMA NONOate) and sodium nitroprusside (SNP), on mucin exocytosis from the human colonic epithelial HT29-Cl.16E cell line. Our results show that MAHMA NONOate and SNP elicited a rapid mucin exocytotic response through a cGMP-dependent and a cGMP-independent pathway respectively. Indeed, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a newly available specific inhibitor of soluble guanylate cyclase, inhibited both cGMP accumulation and subsequent mucin exocytosis evoked by MAHMA NONOate. By contrast, SNP did not alter intracellular cGMP levels, and SNP-mediated mucin exocytosis was not inhibited by ODQ. As expected from two NO donors acting through distinct pathways, the combined action of MAHMA NONOate and SNP led to an additive effect on mucin exocytosis. SNP was likely to act through S-nitrosylation of a cellular target, because cysteine, a reductive thiol that provides decoy targets for SNP through the formation of nitrosocysteine, abolished the early stimulatory effect of SNP on mucin exocytosis. Finally, the fact that in the presence of cysteine SNP was able to trigger a late, ODQ-inhibitable, mucin exocytotic response demonstrates the ability of NO to shift its intracellular signalling pathway depending on the changes of the redox state of the milieu.

A redox-based mechanism for induction of interleukin-1 production by nitric oxide in a human colonic epithelial cell line (HT29-Cl.16E).

We evaluated the effects of two NO donors, sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-1), characterized by alternative redox states, i.e. nitrosonium ion (NO+) and nitric oxide (NO.) respectively, on intracellular interleukin-1 (IL-1) production, by a human colonic epithelial cell line (HT29-Cl.16E). SNP was able to induce intracellular IL-1 alpha production up to 10 h incubation, in a dose-dependent manner. Several experiments provide evidence that the NO+ redox form, and not the free radical NO., is implicated in the IL-1 alpha production: (i) SIN-1, devoid of any NO+ character, led to a very weak IL-1 production as compared with SNP; (ii) the reductive action of a thiol such as cysteine on NO+ led to a dose-dependent increase in NO, concentration, measured as NO2-/NO3- accumulation, and to large decrease in IL-1 production. Dibutyryl cGMP had no effect on IL-1 production, this finding supporting the concept that a cGMP-independent pathway is involved in the intracellular signalling of NO+. Together these results point out that NO, depending on its redox form, is able to modulate IL-1 production in cultured colonic epithelial cells.

Ca2+ and cAMP activate different K+ conductances in the human intestinal goblet cell line HT29-Cl.16E.

The mechanism of regulated Cl- secretion was evaluated in the mucin-secreting cell line HT29-Cl.16E by transepithelial electrophysiology and fura 2 measurements of cytosolic Ca2+. Carbachol by itself was a weak secretagogue, but augmented adenosine 3',5'-cyclic monophosphate (cAMP)-mediated secretion more than twofold, consistent with activation of a rate-limiting K+ conductance. To characterize this conductance, monolayers were apically permeabilized with amphotericin B. At least two types of K+ conductances were identified. One type was activated by elevated cytosolic cAMP levels and inhibited by Ba2+ (inhibitor constant 0.3 mM) in the basolateral solution but was not affected by quinidine or elevated cytosolic Ca2+. The other type was activated by carbachol via cytosolic Ca2+ and was partially inhibited by quinidine (60% inhibition by 2.5 mM quinidine) but was not affected by Ba2+ up to 1 mM. Both conductances appear to be involved in active, transepithelial Cl- secretion in intact monolayers but under different conditions because 1) the cAMP-stimulated short-circuit current (Isc) can be partially inhibited by 1 mM Ba2+ (50%) but not quinidine, 2) the Ba2+ inhibition does not affect the carbachol-induced increase in Isc in cells with elevated cAMP levels, and 3) the carbachol-dependent Isc can be inhibited by quinidine. Therefore, the contribution of the cAMP-dependent K+ conductance appears important for maintaining the membrane potential and therewith Cl- secretion when cAMP is the only messenger for secretion signals, whereas the Ca(2+)-dependent K+ conductance is responsible for the carbachol-stimulated increase in Isc.

Cyclic AMP-induced mucin exocytosis is independent of Cl- movements in human colonic epithelial cells (HT29-Cl.16E).

The human colonic epithelial goblet cell line HT29-Cl.16E was used to test whether stimulated Cl- transport is involved in the mucin exocytotic response to an increase in intracellular cyclic AMP by measuring in parallel the short-circuit current (Isc) and mucin exocytosis. Addition of 50 microM forskolin to HT29-Cl.16E cells resulted in a 2-fold stimulation of mucin release and an increase in Isc by 20 microA/cm2. To evaluate the requirement for cosecretion of Cl-, the Cl- flux was altered by three different manipulations: (1) Cl- in the medium was replaced by the poorly transported anion gluconate; (2) basolateral Cl- influx through the Na(+)-K(+)-2Cl- cotransporter was inhibited by bumetanide; and (3) an inward Cl- flux through the apical plasma membrane was generated by reversing the Cl- gradient. These manipulations did not change the forskolin-stimulated mucin release and thereby provide evidence that Cl- movements are not required for fusion of mucin granules with the plasma membrane.

Role of calcium in carbachol- and neurotensin-induced mucin exocytosis in a human colonic goblet cell line and cross-talk with the cyclic AMP pathway.

The mechanisms of Ca(2+)-induced mucin secretion were examined in monolayers of the differentiated epithelial colon cell line C1.16E by combined measurements of free intracellular Ca2+ ([Ca2+]i) using a fluorescence indicator and mucous secretion using a specific and sensitive electrophoretic assay. Carbachol, a cholinergic agonist, induced an initial concentration-dependent [Ca2+]i peak increasing from 129 +/- 3 nM (basal [Ca2+]i) to 608 +/- 101 nM at 1 x 10(-4) M carbachol with an ED50 of 7 microM, and this was followed by a lower-level plateau. These biphasic effects were reversed by the muscarinic-receptor antagonist atropine. In the absence of extracellular Ca2+, the initial [Ca2+]i peak was maintained while the sustained plateau was abolished. The regulatory peptide neurotensin caused a monophasic transient rise in [Ca2+]i followed by a very rapid return to baseline. The neurotensin-induced rise in [Ca2+]i was concentration-dependent with an ED50 of 4 nM, and was maximal at 1 x 10(-6) M (598 +/- 127 nM). The [Ca2+]i response to neurotensin was not significantly affected by extracellular Ca2+ depletion. Carbachol-induced mucin exocytosis was concentration-dependent with an ED50 of 15 microM, and was inhibited by 35% upon removal of extracellular Ca2+. Neurotensin caused a concentration-dependent rise in mucous secretion with an ED50 of 36 nM, not significantly affected upon removal of extracellular Ca2+. Together our results suggest that while the mucin secretory response to carbachol depends on both the release of Ca2+ from intracellular stores and a Ca2+ influx from external medium, the secretory response to neurotensin is based solely on intracellular Ca2+ mobilization. Finally, evaluation of the cross-talk between the cyclic AMP pathway stimulated by vasoactive intestinal peptide (VIP) and the Ca2+ pathway stimulated by neurotensin or carbachol led to the conclusion that the potentiated secretory response elicited by the combined action of carbachol and VIP requires extracellular Ca2+.

Interferon-gamma modulates cAMP-induced mucin exocytosis without affecting mucin gene expression in a human colonic goblet cell line.

The regulation of intestinal mucin secretion by cytokines, soluble factors released by mucosal activated immune cells, is so far unknown. The aim of the present study was (1) to investigate the regulatory effects of interferon-gamma on baseline and stimulated mucin secretion elicited by an increase in intracellular cAMP, either a short-term increase (induced by vasoactive intestinal peptide or by forskolin) or a long-term increase (cholera toxin-induced), and (2) to attempt to delineate the site of action of interferon-gamma. The in vitro model used was the human colonic goblet cell line Cl.16E, which has already been shown to respond to physiological secretagogues in terms of mucin secretion. We examined the effects of interferon-gamma 1) on mucin exocytosis, measured as release of [3H]glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels, and 2) on mucin biosynthesis, examined at the RNA level using a cDNA probe directed to the MUC2 mucin gene. We demonstrated that, while interferon-gamma did not alter baseline Cl.16E mucin secretion and MUC2 gene expression, it strongly inhibited the protein kinase A-dependent secretory response to VIP, forskolin, or cholera toxin. However, interferon-gamma had no effect on the protein kinase A-dependent MUC2 over-expression induced by cholera toxin. We thus concluded that the target for interferon-gamma inhibition of cAMP-stimulated Cl.16E mucin secretion is distal to protein kinase A and might be a component of the exocytotic machinery. Together, our results establish interferon-gamma as a pharmacologically powerful tool to specifically inhibit stimulated secretory processes without affecting baseline secretion.

Polarised interleukin 8 secretion by HT 29/19A cells.

Interleukin 8 is a neutrophil chemotactic and stimulating cytokine induced by various inflammatory stimuli, including tumour necrosis factor, interleukin 1, and endotoxin. The ability of HT 29/19A enterocytes to synthesise interleukin 8 was studied. The results show that interleukin 1 is an important stimulus for interleukin 8 synthesis and secretion by HT 29/19A cells, being more potent than tumour necrosis factor. The tumour necrosis factor and interleukin 1 induced interleukin 8 secretion by HT 29/19A cells was seen to be polarised according to the direction of stimulation. These results support the concept that mucosal cells (enterocytes) may play an important part in initiating mucosal inflammation. Furthermore, it is proposed that HT 29/19A cells constitute a tool to study stimulus directed polarised cytokine secretion.