Effects of azathioprine and its metabolites on repair mechanisms of the intestinal epithelium in vitro

Erosions and ulcerations of the intestinal epithelium are hallmarks of inflammatory bowel diseases (IBD). Intestinal epithelial cell migration (restitution) and proliferation are pivotal mechanisms for healing of epithelial defects after mucosal injury. In addition, the rate of apoptosis of epithelial cells may modulate intestinal wound healing. The purine antagonists azathioprine (AZA) and 6-mercaptopurine (6-MP) are widely used drugs in the treatment of IBD. In the present study, the hitherto unknown effects of AZA as well as its metabolites 6-MP and 6-thioguanine (6-TG) on repair mechanisms and apoptosis of intestinal epithelia were analysed. Intestinal epithelial cell lines (human Caco-2, T-84 and HT-29 cells, rat IEC-6 cells) were incubated with AZA, 6-MP or 6-TG for 24 h (final concentrations 0.1-10 microM). Migration of Caco-2 and IEC-6 cells was analysed by in vitro restitution assays. Caco-2 and IEC-6 cell proliferation was evaluated by measurement of [3H]thymidine incorporation into DNA. Apoptosis of Caco-2, T-84, HT-29 and IEC-6 cells was assessed by histone ELISA, 4'6'diamidino-2'phenylindole-dihydrochloride staining as well as flow cytometric analysis of Annexin V/propidium iodide (PI)-stained cells. Cell cycle progression was evaluated by PI staining and flow cytometry. Epithelial restitution was not significantly affected by any of the substances tested. However, proliferation of intestinal epithelial cells was inhibited in a dose-dependent manner (maximal effect 92%) by AZA, 6-MP as well as 6-TG. In HT-29 cells, purine antagonist-effected inhibition of cell proliferation was explained by a cell cycle arrest in the G2 phase. In contrast, AZA, 6-MP and 6-TG induced no cell cycle arrest in Caco-2, T-84 and IEC-6 cells. AZA, 6-MP as well as 6-TG induced apoptosis in the non-transformed IEC-6 cell line but not in human Caco-2, T-84 and HT-29 cells. In summary, AZA and its metabolites exert no significant effect on intestinal epithelial restitution. However, they profoundly inhibit intestinal epithelial cell growth via various mechanisms: they cause a G2 cell cycle arrest in HT-29 cells, induce apoptosis in IEC-6 cells and dose-dependently inhibit intestinal epithelial proliferation.     

Regulation of 11 beta-hydroxysteroid dehydrogenase enzymes in the rat kidney by estradiol

The 11beta-hydroxysteroid dehydrogenase (11betaHSD) type 1 (11betaHSD1) enzyme is an NADP+-dependent oxidoreductase, usually reductase, of major glucocorticoids. The NAD+-dependent type 2 (11betaHSD2) enzyme is an oxidase that inactivates cortisol and corticosterone, conferring extrinsic specificity of the mineralocorticoid receptor for aldosterone. We reported that addition of a reducing agent to renal homogenates results in the monomerization of 11betaHSD2 dimers and a significant increase in NAD+-dependent corticosterone conversion. Estrogenic effects on expression, dimerization, and activity of the kidney 11betaHSD1 and -2 enzymes are described herein. Renal 11betaHSD1 mRNA and protein expressions were decreased to very low levels by estradiol (E2) treatment of both intact and castrated male rats; testosterone had no effect. NADP+-dependent enzymatic activity of renal homogenates from E2-treated rats measured under nonreducing conditions was less than that of homogenates from intact animals. Addition of 10 mM DTT to aliquots from these same homogenates abrogated the difference in NADP+-dependent activity between E2-treated and control rats. In contrast, 11betaHSD2 mRNA and protein expressions were significantly increased by E2 treatment. There was a marked increase in the number of juxtamedullary proximal tubules stained by the antibody against 11betaHSD2 after the administration of E2. Notwithstanding, neither the total corticosterone and 11-dehydrocorticosterone excreted in the urine nor their ratio differed between E2- and vehicle-treated rats. NAD+-dependent enzymatic activity in the absence or presence of a reducing agent demonstrated that the increase in 11betaHSD2 protein was not associated with an increase in in vitro activity unless the dimers were reduced to monomers.     

Glucocorticoid metabolism and Na+ transport in chicken intestine

The role of aldosterone in regulation of electrogenic Na+ transport is well established, though mineralocorticoid receptors bind glucocorticoids with similar binding affinity as aldosterone and plasma concentration of aldosterone is much lower than glucocorticoids. In mammals, the aldosterone specificity is conferred on the low-selective mineralocorticoid receptors by glucocorticoid inactivating enzyme 11beta-hydroxysteroid dehydrogenase (11HSD) that converts cortisol or corticosterone into metabolites (cortisone, 11-dehydrocorticosterone) with lower affinity for these receptors. The present study examined the chicken intestine, whether changes in 11HSD activity are able to modulate the effect of corticosterone on Na+ transport, and how the metabolism of this hormone is distributed within the intestinal wall. This study shows that not only aldosterone, but also corticosterone (B), was able to increase the electrogenic Na+ transport in chicken caecum in vitro. The effect of corticosterone was higher in the presence of carbenoxolone, an inhibitor of steroid dehydrogenases, and was comparable to the effect of aldosterone. The metabolism of B in the intestine was studied; results showed oxidation of this steroid to 11-dehydrocorticosterone (A) and reduction to 11-dehydro-20beta-dihydrocorticosterone (20diA) as the main metabolic products at low nanomolar concentration of the substrate. In contrast, 20beta-dihydrocorticosterone and 20diA were the major products at micromolar concentration of B. Progesterone was converted to 20beta-dihydroprogesterone. The metabolism of corticosterone was localized predominantly in the intestinal mucosa (enterocytes). In conclusion, the oxidation at position C11 and reduction at position C20 suggest that both 11HSD and 20beta-hydroxysteroid dehydrogenase (20HSD) operate in the chicken intestine and that the mucosa of avian intestine possesses a partly different system of modulation of corticosteroid signals than mammals. This system seems to protect the aldosterone target tissue against excessive concentration of corticosterone and progesterone.     

Novel nuclear corticosteroid binding in rat small intestinal epithelia

When small intestinal epithelial cells are incubated with [(3)H]corticosterone, nuclear binding is displaced neither by aldosterone nor RU-28362, suggesting that [(3)H]corticosterone is binding to a site distinct from mineralocorticoid receptor and glucocorticoid receptor. Saturation and Scatchard analysis of nuclear [(3)H]corticosterone binding demonstrate a single saturable binding site with a relatively low affinity (49 nM) and high capacity (5 fmol/microg DNA). Competitive binding assays indicate that this site has a unique steroid binding specificity, which distinguishes it from other steroid receptors. Steroid specificity of nuclear binding mirrors inhibition of the low 11beta-dehydrogenase activity, suggesting that binding may be to an 11beta-hydroxysteroid dehydrogenase (11betaHSD) isoform, although 11betaHSD1 is not present in small intestinal epithelia and 11betaHSD2 does not colocalize intracellularly with the binding site. In summary, a nuclear [(3)H]corticosterone binding site is present in small intestinal epithelia that is distinct from other steroid receptors and shares steroid specificity characteristics with 11betaHSD2 but is distinguishable from the latter by its distinct intracellular localization.     

Rapid mechanisms of glucocorticoid signaling in the Leydig cell

Stress-mediated elevations in circulating glucocorticoid levels lead to corresponding rapid declines in testosterone production by Leydig cells in the testis. In previous studies we have established that glucocorticoids act on Leydig cells directly, through the classic glucocorticoid receptor (GR), and that access to the GR is controlled prior to the GR by a metabolizing pathway mediated by the type 1 isoform of 11beta-hydroxysteroid dehydrogenase (11betaHSD1). This enzyme is bidirectional (with both oxidase and reductase activities) and in the rat testis is exclusively localized in Leydig cells where it is abundantly expressed and may catalyze the oxidative inactivation of glucocorticoids. The predominant reductase direction of 11betaHSD1 activity in liver cells is determined by an enzyme, hexose-6-phosphate dehydrogenase (H6PDH), on the luminal side of the smooth endoplasmic reticulum (SER). Generation of the pyridine nucleotide cofactor NADPH by H6PDH stimulates the reductase direction of 11betaHSD1 resulting in increased levels of active glucocorticoids in liver cells. Unlike liver cells, steroidogenic enzymes including 17beta-hydroxysteroid dehydrogenase 3 (17betaHSD3) forms the coupling with 11betaHSD1. Thus the physiological concentrations of androstenedione serve as a substrate for 17betaHSD3 utilizing NADPH to generate NADP+, which drives 11betaHSD1 in Leydig cells primarily as an oxidase; thus eliminating the adverse effects of glucocorticoids on testosterone production. At the same time 11betaHSD1 generates NADPH which promotes testosterone biosynthesis by stimulating 17betaHSD3 in a cooperative cycle. This enzymatic coupling constitutes a rapid mechanism for modulating glucocorticoid control of testosterone biosynthesis. Under stress conditions, glucocorticoids also have rapid actions to suppress cAMP formation thus to lower testosterone production.     

Local amplification of glucocorticoids by 11 beta-hydroxysteroid dehydrogenase type 1 promotes macrophage phagocytosis of apoptotic leukocytes

Glucocorticoids promote macrophage phagocytosis of leukocytes undergoing apoptosis. Prereceptor metabolism of glucocorticoids by 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) modulates cellular steroid action. 11beta-HSD type 1 amplifies intracellular levels of active glucocorticoids in mice by reactivating corticosterone from inert 11-dehydrocorticosterone in cells expressing the enzyme. In this study we describe the rapid (within 3 h) induction of 11beta-HSD activity in cells elicited in the peritoneum by a single thioglycolate injection in mice. Levels remained high in peritoneal cells until resolution. In vitro experiments on mouse macrophages demonstrated that treatment with inert 11-dehydrocorticosterone for 24 h increased phagocytosis of apoptotic neutrophils to the same extent as corticosterone. This effect was dependent upon 11beta-HSD1, as 11beta-HSD1 mRNA, but not 11beta-HSD2 mRNA, was expressed in these cells; 11-dehydrocorticosterone was ineffective in promoting phagocytosis by Hsd11b1(-/-) macrophages, and carbenoxolone, an 11beta-HSD inhibitor, prevented the increase in phagocytosis elicited in wild-type macrophages by 11-dehydrocorticosterone. Importantly, as experimental peritonitis progressed, clearance of apoptotic neutrophils was delayed in Hsd11b1(-/-) mice. These data point to an early role for 11beta-HSD1 in promoting the rapid clearance of apoptotic cells during the resolution of inflammation and indicate a novel target for therapy.     

Epithelial and adipose cells isolated from mammary glands of pregnant and lactating rats differ in 11 beta-hydroxysteroid dehydrogenase activity

Both adipose and epithelial cells isolated from the mammary glands of pregnant and lactating rats show 11 beta-hydroxysteroid dehydrogenase (11-HSD) activity, as measured by conversion of corticosterone to 11-dehydrocorticosterone. Activity in adipose cells from pregnant rats is 3-fold higher than in lactating rats. Epithelial cells from pregnant rats show one-twentieth of the activity of adipose cells, and activity is lower still in epithelial cells from lactating rats. Explants incubated for 48 h extensively metabolized corticosterone to 11-dehydrocorticosterone, and to a much lesser extent to a second unknown metabolite which is found in tissue extracts but not conditioned medium. Mammary gland 11-HSD may thus constitute one of the physiological mechanisms preventing premature milk production in response to glucocorticoids.     

CXCL8 modulates human intestinal epithelial cells through a CXCR1 dependent pathway

BACKGROUND: CXCL8 (previously known as Interleukin-8), a member of the alpha-chemokine family of chemotactic cytokines, stimulates intestinal neutrophil activation and chemotaxis. As intestinal epithelial cells have been recently shown to produce CXCL8, the aim of this study was to identify functional activities of CXCL8 on intestinal epithelial cells. METHODS: The expression of CXCL8 receptors CXCR1 and CXCR2 was assessed by RT-PCR and FACS analysis in human Caco-2 and HT-29 cells. The effects of CXCL8 on intestinal epithelial proliferation were assessed with colorimetric MTT assays and the effects on epithelial restitution with an in vitro migration model using Caco-2 and HT-29 cells. RESULTS: While the expression of both CXCR1 mRNA and protein could be demonstrated by RT-PCR and FACS analysis in human Caco-2 and HT-29 cells, no expression of CXCR2 was observed in these cell lines. Colorimetric MTT assays revealed that CXCL8 does not modulate cell proliferation in HT-29 and Caco-2 cells. In contrast, CXCL8 significantly enhanced intestinal epithelial migration in an in vitro migration model of HT-29 and Caco-2 cells. Enhancement of intestinal epithelial cell migration by CXCL8 was partially CXCR1-dependent and TGFbeta-independent. CONCLUSION: CXCL8 exerts functional effects on intestinal epithelial cells that may be relevant for intestinal inflammation and mucosal healing.     

Renal 11-beta-hydroxysteroid dehydrogenase: a mechanism ensuring mineralocorticoid specificity

In vitro studies with mineralocorticoid receptors (MR) have shown that they are non-specific and do not distinguish between glucocorticoids (cortisol in man, corticosterone in rodents) and aldosterone. These findings contrast with in vivo aldosterone selectivity. Our studies on the congenital deficiency of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD; which converts cortisol to cortisone or corticosterone to 11-dehydrocorticosterone) and acquired deficiency secondary to liquorice or carbenoxolone indicate that this enzyme plays a crucial role in protecting the MR from glucocorticoid exposure. The localisation of 11 beta-OHSD in both the proximal and distal nephron suggests that it has both an autocrine and a paracrine role. The presence of this protective mechanism in the toad bladder suggests that it is at least 300 million years old.     

Cellular isoform of the prion protein PrPc in human intestinal cell lines: genetic polymorphism at codon 129, mRNA quantification and protein detection in lipid rafts

The cellular isoform of the normal prion protein PrP(c), encoded by the PRNP gene, is expressed in human intestinal epithelial cells where it may represent a potential target for infectious prions. We have sequenced the PRNP gene in Caco-2 and HT-29 parental and clonal cell lines, and found that these cells have a distinct polymorphism at codon 129. HT-29 cells are homozygous Met/Met, whereas Caco-2 cells are heterozygous Met/Val. The 129Val variant was also detected in Caco-2 mRNAs. Real-time PCR quantifications revealed that PrP(c) mRNAs were more expressed in HT-29 cells than in Caco-2 cells. These data were confirmed by studying the expression of PrP(c) in plasma membranes and lipid rafts prepared from these cells. Overall, these results may be important in view of using human intestinal cell lines Caco-2 and HT-29 as cellular in vitro models to study the initial steps of prion propagation after oral inoculation.     

11-Beta hydroxysteroid dehydrogenase type 2 expression in white adipose tissue is strongly correlated with adiposity

Glucocorticoid action within the cells is regulated by the levels of glucocorticoid receptor (GR) expression and two enzymes, 11-beta hydroxysteroid dehydrogenase type 1 (11betaHSD1), which converts inactive to active glucocorticoids, and 11-beta hydroxysteroid dehydrogenase type 2 (11betaHSD2), which regulates the access of active glucocorticoids to the receptor by converting cortisol/corticosterone to the glucocorticoid-inactive form cortisone/dehydrocorticosterone. Male Wistar rats developed obesity by being fed a high-fat diet for 56 days, and GR, 11betaHSD1 and 11betaHSD2 gene expression were compared with control-diet fed animals. Gene expression analysis of 11betaHSD1, 11betaHSD2 and GR were performed by RT-PCR in subcutaneous and retroperitoneal adipose tissue. High-fat fed animals overexpressed 11betaHSD2 in subcutaneous but not in retroperitoneal fat. Interestingly, mRNA levels strongly correlated in both tissues with different parameters related to obesity, such as body weight, adiposity and insulin resistance, suggesting that this gene is a reliable marker of adiposity in this rat model of obesity. Thus, 11betaHSD2 is expressed in adipose tissue by both adipocytes and stromal-vascular cells, which suggests that this enzyme may play an important role in preventing fat accumulation in adipose tissue.     

Upregulation of activin A gene by butyrate in human colon cancer cell lines

Activin A has been reported to play a role in the progression of colorectal cancer. Because dietary fiber protects against colorectal cancer, we hypothesized that butyrate, a fermentation product of dietary fiber, may affect the expression of activin A in colon cancer cells. Semiquantitative RT-PCR demonstrated that the activin A gene was upregulated by sodium butyrate in the human colon cancer cell lines HT-29 and Caco-2 in a concentration- and time-dependent manner. However, the activin A gene did not respond to sodium butyrate in the human normal colonic cell line FHC, rat normal intestinal epithelial cell (IEC) line IEC-6, and the explant of rat colon. Flow cytometry and agarose gel electrophoresis of genomic DNA revealed that cell cycle arrest and apoptosis were induced by sodium butyrate but not exogenous activin A in HT-29 cells, indicating that activin A could not act as an autocrine factor in colon cancer cells. By assuming that activin A promotes colorectal cancer spread as a paracrine factor, our findings suggest that butyrate could act as a tumor promoter in some circumstances.     

Focal adhesion kinase protein levels in gut epithelial motility

Mucosal healing requires migration and proliferation. Most studies of focal adhesion kinase (FAK), a protein that regulates motility, proliferation, and apoptosis, have focused on rapid phosphorylation. We reported lower FAK protein levels in motile Caco-2 colon cancer cells and postulated that this reduction in FAK available for activation might impact cell migration and mucosal healing. Therefore, total and active FAK (FAK(397)) immunoreactivity was assessed at the migrating fronts of human Caco-2 and rat IEC-6 intestinal epithelial cells. Caco-2 and IEC-6 motility, quantitated as migration into linear or circular wounds, was examined following FAK protein inhibition by small interfering RNA (siRNA). FAK protein stability and mRNA expression were ascertained by cycloheximide decay, RT-PCR, and in situ hybridization in static and migrating Caco-2 cells. Cells at the migrating front of Caco-2 and IEC-6 monolayers exhibited lower immunostaining for both total and activated FAK than cells immediately behind the front. Western blot analysis also demonstrated diminished FAK protein levels in motile cells by >/=30% in both the differential density seeding and multiple scrape models. siRNA FAK protein inhibition enhanced motility in both the linear scrape (20% in Caco-2) and circular wound (16% in Caco-2 and 19% in IEC-6 cells) models. FAK protein degradation did not differ in motile and static Caco-2 cells and was unaffected by FAK(397) phosphorylation, but FAK mRNA was lower in migrating Caco-2 cells. Thus FAK protein abundance appears regulated at the mRNA level during gut epithelial cell motility and may influence epithelial cell migration coordinately with signals that modify FAK phosphorylation.     

Glucocorticoid metabolism by 11-beta hydroxysteroid dehydrogenase type 2 modulates human mineralocorticoid receptor transactivation activity

The mineralocorticoid receptor (MR) binds aldosterone, but also glucocorticoid hormones (corticosterone in rodents, cortisol in humans), which largely prevail in the plasma. To prevent permanent and maximal occupancy of MR by glucocorticoid hormones in aldosterone-target cells, specific effects of aldosterone require metabolism of glucocorticoid hormones into 11-dehydroderivatives by 11-beta hydroxysteroid dehydrogenase (11-HSD2). We analyzed the effect of corticosterone or 11-dehydrocorticosterone (11-DHC) on the transactivation activity of the MR, transiently expressed in a new renal cell line expressing 11-HSD2. We show that, because of its metabolism by 11-HSD2, corticosterone is a poor activator of MR transactivation, except at micromolar concentrations, where the enzyme is saturated. We also show that high micromolar concentrations of 11 DHC are required to activate the MR. The weak antagonist property of 11-DHC on aldosterone-induced hMR transactivations is also documented. Such partial agonist activity of 11-DHC is discussed in the light of its positioning in a three-dimensional model of the MR ligand-binding domain.     

Molecular and cellular effects of cis-9, trans-11-conjugated linoleic acid in enterocytes: effects on proliferation, differentiation, and gene expression

It has been hypothesized that dietary conjugated linoleic acids (CLA) may inhibit colon tumorigenesis. The aim of our study was to investigate the cellular and molecular effects of cis-9 (9Z), trans-11 (11E)-CLA on the proliferation, differentiation, interaction with peroxisome proliferator-activated receptors (PPARs), and expression of genes relevant in the APC-beta-catenin-TCF4 signalling pathway in human HT-29 and Caco-2 colon cells. We found that 9Z,11E-CLA inhibited the proliferation of HT-29 and Caco-2 cells. Trans-vaccenic acid (VA) showed no antiproliferative effects at all. We determined that 9Z,11E-CLA induced cell differentiation as measured by intestinal alkaline phosphatase (IAP) enzyme activity in Caco-2 cells, mRNA expression of IAP, and activation of a 5' flanking region of IAP. The 9Z,11E-CLA activated human PPARdelta as measured in a reporter gene assay. Treatment of HT29 cells in the poliferation phase with 9Z,11E-CLA repressed mRNA-expression of proliferation genes such as c-myc, cyclin D1 and c-jun in a concentration dependent manner. The promoter activities of c-myc and AP1 were also inhibited after incubation with 9Z,11E-CLA. beta-Catenin mRNA and protein expression was also repressed by the treatment with 9Z,11E-CLA. In addition, the mRNA expression of PPARdelta was repressed by treatment of the HT-29 cells with 9Z,11E-CLA. We conclude that 9Z,11E-CLA has an antiproliferative effect at the cellular and molecular levels in human colon cells. The results indicate that the preventive effects of CLA in the development of colon cancer may be due to their downregulation of some target genes of the APC-beta-catenin-TCF-4- and PPARdelta signalling pathway.     

NF-kappaB-inducing kinase restores defective IkappaB kinase activity and NF-kappaB signaling in intestinal epithelial cells

Cytokine-stimulated IkappaBalpha degradation is impaired in HT-29 and primary intestinal epithelial cells. To gain more insight into the mechanism of this defect, we dissected cytokine-induced NF-kappaB signaling pathway in HT-29 cells. IL-1beta and TNF, alone or in combination with IFNgamma, failed to induce IkappaBalpha or IkappaBbeta degradation in HT-29 cells. Despite similar 125I-IL-1beta binding, HT-29 cells displayed no IRAK degradation, a 75% reduction of IKK activity, and decreased IkappaBalpha phosphorylation, NF-kappaB DNA binding activity and IL-8 mRNA accumulation in response to IL-1beta compared to Caco-2 cells. Selective activation of NF-kappaB pathway by adenoviral delivery of NF-kappaB-inducing kinase (Ad5NIK) or IKKbeta (Ad5IKKbeta) strongly activated IKK activity (>20 fold) in HT-29 cells with concomitant endogenous IkappaBalpha serine 32 phosphorylation and total IkappaBalpha degradation. In addition, NF-kappaB DNA binding activity and IL-8 secretion is higher in Ad5NIK-infected than in IL-1beta-stimulated HT-29 cells. These data show that altered NF-kappaB signaling is associated with impaired stimulation of an upstream IKK activator.     

Differential glucocorticoid effects on repair mechanisms and NF-kappaB activity in the intestinal epithelium

Glucocorticoids are effective agents in the management of inflammatory bowel diseases. However, information about their effects on repair mechanisms of the intestinal epithelium is incomplete.Therefore, the aim was to analyse in vitro effects of glucocorticoids on proliferation, restitution, and apoptosis as well as their effects on activity and expression of nuclear factor (NF)-kappaB, a known regulator of apoptosis and inflammation, in intestinal epithelial cells.Non-transformed rat jejunum epithelial cells (IEC-6) were cultured in the presence and absence of various concentrations of prednisolone and budesonide. IEC-6 cell proliferation was assessed by [3H]-thymidine incorporation. Restitution was analysed by an IEC-6 in vitro assay. Apoptosis was evaluated by ELISA and fluorescence microscopy. DNA binding activity and nuclear expression of NF-kappaB was determined by electrophoretic mobility shift assays and Western blotting, respectively.Prednisolone and budesonide stimulated IEC-6 cell proliferation at low to medium pharmacologic concentrations (prednisolone: 10(-9) to 10(-6) M; budesonide: 10(-11) to 10(-8) M). In contrast, high concentrations (>5 x 10(-5) M) had inhibitory effects on proliferation. 10(-7) M prednisolone and 10(-8) M budesonide increased restitution of IEC-6 cells, whereas high concentrations (10(-4) M) of prednisolone and budesonide decreased restitution. Apoptosis of IEC-6 cells was substantially enhanced by 10(-4) M budesonide; apoptosis was slightly increased by the highest prednisolone concentration used (5 x 10(-4) M). Furthermore, both glucocorticoids inhibited DNA binding activity and nuclear NF-kappaB expression in IEC-6 cells in a dose- and time-dependent fashion.In conclusion, prednisolone and budesonide modulate repair mechanisms of intestinal epithelial cells in vitro in a dose-dependent manner and profoundly modulate the inflammatory regulator NF-kappaB.