Escherichia coli Shiga toxins induce apoptosis in epithelial cells that is regulated by the Bcl-2 family

Human intestinal cells lack globotriaosylceramide (Gb(3)), the receptor for Shiga toxin-1 (Stx1) and Shiga toxin-2 (Stx2). Therefore, the role of these toxins in mediating intestinal disease during infection with Shiga toxin-producing Escherichia coli is unclear. The aims of this study were to determine whether Stx1 and Stx2 induce apoptosis in epithelial cells expressing (HEp-2, Caco-2) or lacking (T84) Gb(3) and to characterize the role of the Bcl-2 family. Stx1 (12.5 ng/ml) induced apoptosis in both HEp-2 (21.9 +/- 7.9% vs. 0.8 +/- 0.3%, P = 0.01) and Caco-2 (10.1 +/- 1.2% vs. 3.1 +/- 0.4%, P = 0.006) cells but not in Gb(3)-deficient T84 cells. Toxin-mediated apoptosis of HEp-2 cells was associated with enhanced expression of the proapoptotic protein Bax. Inhibition of caspase activation prevented toxin-stimulated apoptosis. In addition, overexpression of Bcl-2 by transient transfection blocked Stx1-stimulated cell death. These findings indicate that Shiga toxins produced by E. coli signal Gb(3)-expressing epithelial cells to undergo apoptosis in association with enhanced Bax expression, thereby resulting in activation of the caspase cascade.     

Latrunculin B facilitates Shiga toxin 1 transcellular transcytosis across T84 intestinal epithelial cells

Shiga toxins (Stx), released into the intestinal lumen by enterohemorrhagic Escherichia coli (EHEC), are major virulence factors responsible for gastrointestinal and systemic illnesses. These pathologies are believed to be due to the action of the toxins on endothelial cells, which express the Stx receptor, the glycosphingolipid Gb3. To reach the endothelial cells, Stx must translocate across the intestinal epithelial monolayer. This process is poorly understood. We investigated Stx1 movement across the intestinal epithelial T84 cell model and the role of actin turnover in this transcytosis. We showed that changes in the actin cytoskeleton due to latrunculin B, but not cytochalasin D or jasplakinolide, significantly facilitate toxin transcytosis across T84 monolayers. This trafficking is transcellular and completely inhibited by tannic acid, a cell impermeable plasma membrane fixative. This indicates that actin turnover could play an important role in Stx1 transcellular transcytosis across intestinal epithelium in vitro. Since EHEC attachment to epithelial cells causes an actin rearrangement, this finding may be highly relevant to Stx-induced disease.     

Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo

Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) is a clinically important foodborne pathogen that colonizes human colon epithelium and induces acute colonic inflammation, but does not invade the epithelial cells. Whereas Shiga toxin (Stx) and bacterial flagellin have been studied for their ability to upregulate the production of proinflammatory chemokines by cultured human colon cancer cell lines, the relevance of studies in colon cancer cell lines to the production of proinflammatory signals by normal epithelial cells in EHEC-infected human colon is not known. We show herein that Stx does not bind to human colon epithelium in vivo. Moreover, globotriaosylceramide (Gb3/CD77) synthase, the enzyme required for synthesis of the Gb3/CD77 receptor for Stx, was not expressed by normal or inflamed human colon epithelium in vivo. In contrast, Toll-like receptor (TLR) 5, the receptor for bacterial flagellin, was expressed by normal human colon epithelium and by colon epithelium in human intestinal xenografts. EHEC H7 flagellin instilled in the lumen of human colon xenografts that contain an intact human epithelium upregulated the expression of epithelial cell proinflammatory chemokines, which was accompanied by a subepithelial influx of neutrophils. Isogenic mutants of EHEC that lacked flagellin did not significantly upregulate prototypic neutrophil and dendritic cell chemoattractants by model human colon epithelia, irrespective of Stx production. We conclude that EHEC H7 flagellin and not Stx is the major EHEC factor that directly upregulates proinflammatory chemokine production by human colon epithelium in vivo.     

An enteroaggregative Escherichia coli strain of serotype O111:H12 damages and invades cultured T84 cells and human colonic mucosa

The pathogenic mechanisms of enteroaggregative Escherichia coli (EAEC) are not well defined. We investigated the interaction of EAEC strain 236 (serotype O111:H12) with polarised Caco-2 and T84 human intestinal epithelial cells lines, and with human jejunal and colonic mucosa. Strain 236 adhered to both polarised cell lines and to both intestinal tissue types, but caused severe damage and was invasive only in T84 cells and colonic mucosa. In contrast, prototype EAEC strain 042, which also adhered to the cultured intestinal cell lines, did not adhere to or invade jejunal or colonic tissue. These observations suggest a heterogeneity of virulence properties within the EAEC category of diarrhoea-causing E. coli.     

Proteasome modulating agents induce rAAV2-mediated transgene expression in human intestinal epithelial cells

Intestinal gene transfer offers promise as a therapeutic option for treatment of both intestinal and non-intestinal diseases. Recombinant adeno-associated virus serotype 2, rAAV2, based vectors have been utilized to transduce lung epithelial cells in culture and in human subjects. rAAV2 transduction of intestinal epithelial cells, however, is limited both in culture and in vivo. Proteasome-inhibiting agents have recently been shown to enhance rAAV2-mediated transgene expression in airway epithelial cells. We hypothesized that similar inhibition of proteasome-related cellular processes can function to induce rAAV2 transduction of intestinal epithelial cells. Our results demonstrate that combined treatment with proteasome-modulating agents MG101 (N-acetyl-L-leucyl-L-leucyl-L-norleucine) and Doxorubicin synergistically induces rAAV2-mediated luciferase transgene expression by >400-fold in undifferentiated Caco-2 cells. In differentiated Caco-2 monolayers, treatment with MG101 and Doxorubicin induces transduction preferentially from the basolateral cell surface. In addition to Caco-2 cells, treatment with MG101 and Doxorubicin also results in enhanced rAAV2 transduction of HT-29, T84, and HCT-116 human intestinal epithelial cell lines. We conclude that MG101 and Doxorubicin mediate generic effects on intestinal epithelial cells that result in enhanced rAAV2 transduction. Use of proteasome-modulating agents to enhance viral transduction may facilitate the development of more efficient intestinal gene transfer protocols.     

SDF-1/CXCL12 regulates cAMP production and ion transport in intestinal epithelial cells via CXCR4

Human colonic epithelial cells express CXCR4, the sole cognate receptor for the chemokine stromal cell-derived factor (SDF)-1/CXC chemokine ligand (CXCL) 12. The aim of this study was to define the mechanism and functional consequences of signaling intestinal epithelial cells through the CXCR4 chemokine receptor. CXCR4, but not SDF-1/CXCL12, was constitutively expressed by T84, HT-29, HT-29/-18C1, and Caco-2 human colon epithelial cell lines. Studies using T84 cells showed that CXCR4 was G protein-coupled in intestinal epithelial cells. Moreover, stimulation of T84 cells with SDF-1/CXCL12 inhibited cAMP production in response to the adenylyl cyclase activator forskolin, and this inhibition was abrogated by either anti-CXCR4 antibody or receptor desensitization. Studies with pertussis toxin suggested that SDF-1/CXCL12 activated negative regulation of cAMP production through G(i)alpha subunits coupled to CXCR4. Consistent with the inhibition of forskolin-stimulated cAMP production, SDF-1/CXCL12 also inhibited forskolin-induced ion transport in voltage-clamped polarized T84 cells. Taken together, these data indicate that epithelial CXCR4 can transduce functional signals in human intestinal epithelial cells that modulate important cAMP-mediated cellular functions.     

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.     

Macrophage-inflammatory protein-3alpha mediates epidermal growth factor receptor transactivation and ERK1/2 MAPK signaling in Caco-2 colonic epithelial cells via metalloproteinase-dependent release of amphiregulin

Previously, we reported that normal colonocytes produce the memory CD4(+) T cell-directed chemokine MIP-3alpha, and that epithelial MIP-3alpha levels are elevated in inflammatory bowel disease. Interestingly, the unique receptor for MIP-3alpha, CCR6, is expressed by a variety of cell types including colonocytes, suggesting that MIP-3alpha may regulate additional biological activities in the intestine. The aim of this study was to determine whether MIP-3alpha can induce intestinal epithelial cell proliferation and to examine the signaling mechanisms that mediate this response. We show that nonstimulated Caco-2 and HT-29 colonic epithelial cells express CCR6, and that stimulation of Caco-2 cells by MIP-3alpha can dose dependently increase cell proliferation as well as activate the epidermal growth factor receptor (EGFR) and ERK1/2 MAPK. MIP-3alpha-mediated ERK1/2 activation in Caco-2 cells appeared to require metalloproteinase-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing polyclonal Ab significantly reduced MIP-3alpha-mediated, but not EGF-mediated Caco-2 cell proliferation. Taken together, our findings indicate that MIP-3alpha can regulate mitogenic signaling in colonic epithelial cells and thus may serve an important homeostatic function in the intestine by regulating tissue turnover and maintenance of the epithelium, in addition to its role in regulating leukocyte recruitment.     

Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway

Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A₁, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.     

Glucagon-like peptide 2 improves intestinal wound healing through induction of epithelial cell migration in vitro-evidence for a TGF--beta-mediated effect

BACKGROUND/AIMS: In vitro studies suggest that glucagon-like peptide 2 (GLP-2), secreted from enteroendocrine cells in the gastrointestinal tract after food intake, is able to ameliorate mucosal injury in settings of human disease characterized by injury and dysfunction of the intestinal mucosal epithelium. We evaluated this potential of GLP-2 after epithelial trauma by using two in vitro models measuring intestinal epithelial cell proliferation and cell migration. MATERIALS AND METHODS: Injuries were induced in confluent monolayers of the small intestinal cells lines IEC-6 and IEC-18, as well as in the colonic cell lines Caco-2 and Colo 320. GLP-2 (50-500 nM) or other peptides were added to the media. Wound healing was investigated after 24 h by quantification of the number of cells migrating across the wound edge. Proliferation of cells was assessed by using photometric mitochondrial incorporation measurement of MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide). Monoclonal TGF-beta antibodies were added to wounded monolayers to examine whether the GLP-2-induced wound healing was TGF-beta-mediated. RESULTS: Migration assessments revealed a significant stimulation of GLP-2-induced migration in IEC-6 and IEC-18 monolayers compared to the placebo group. No effect was observed in the colon cancer cell lines Caco-2 and Colo 320. Results of the proliferation assays show a significant inhibition of proliferation by GLP-2 in small intestinal cell lines whereas a dose-dependent stimulation of proliferation in colonic epithelial cells was observed. Addition of neutralizing TGF-beta1 antibodies to wounded IEC-6 and IEC-18 monolayers incubated with GLP-2 significantly reduced the number of migrating cells to the level of the placebo group. CONCLUSIONS: In our in vitro model, it was shown that the GLP-2-induced improvement of intestinal wound healing is TGF-beta-mediated. These effects were predominant in the epithelium of the small intestine compared to colonic epithelium. Our findings provide further insight into mechanisms leading to GLP-2-induced mucosal wound healing. These results suggest that GLP-2 or analogues of this peptide may potentially be useful for the treatment of intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.     

Induction by activated macrophage-like THP-1 cells of apoptotic and necrotic cell death in intestinal epithelial Caco-2 monolayers via tumor necrosis factor-alpha

Intestinal epithelial cells interact with immune cells located in the intestinal epithelium via soluble factors. An in vitro model system using coculture was constructed to analyze the effect of macrophages on intestinal epithelial cells, and human intestinal epithelial-like Caco-2 monolayers and activated macrophage-like THP-1 cells were used in this study. Coculturing with THP-1 cells resulted in an increase of lactate dehydrogenase release from Caco-2 and a decrease in the transepithelial electrical resistance of the monolayers, showing that coculturing with THP-1 induced cell damage to Caco-2 cells. This disruption was significantly suppressed by adding anti-TNF-alpha antibody and etanercept, strongly suggesting that TNF-alpha secreted from THP-1 had caused cell damage to Caco-2 monolayers. The disrupted Caco-2 monolayers showed both apoptotic and necrotic characteristics by morphological and biochemical analyses. TNFRI and NF-kappaB seem to have been involved in this regulation. It is suggested that this phenomenon is similar in some respects to that observed with IBD and that this in vitro coculture system could be a good model for searching for the drugs or food substances that can be used to treat or prevent IBD.     

Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium

The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1alpha stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-kappaB target gene in the intestinal epithelium as blocking NF-kappaB activation inhibits the up-regulated expression of hBD-2 in response to IL-1alpha stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.     

Normal mouse intestinal epithelial cells as a model for the in vitro invasion of Trichinella spiralis infective larvae

It has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine; however, the mechanisms used by the parasite in the establishment of its intramulticellular niche in the intestine are unknown. Although the previous observations indicated that invasion also occurs in vitro when the infective larvae are inoculated onto cultures of intestinal epithelial cells (e.g., human colonic carcinoma cell line Caco-2, HCT-8), a normal readily manipulated in vitro model has not been established because of difficulties in the culture of primary intestinal epithelial cells (IECs). In this study, we described a normal intestinal epithelial model in which T. spiralis infective larvae were shown to invade the monolayers of normal mouse IECs in vitro. The IECs derived from intestinal crypts of fetal mouse small intestine had the ability to proliferate continuously and express specific cytokeratins as well as intestinal functional cell markers. Furthermore, they were susceptible to invasion by T. spiralis. When inoculated onto the IEC monolayer, infective larvae penetrated cells and migrated through them, leaving trails of damaged cells heavily loaded with T. spiralis larval excretory-secretory (ES) antigens which were recognized by rabbit immune sera on immunofluorescence test. The normal intestinal epithelial model of invasion mimicking the natural environment in vivo will help us to further investigate the process as well as the mechanisms by which T. spiralis establishes its intestinal niche.     

Nucleoside transport in human colonic epithelial cell lines: evidence for two Na+-independent transport systems in T84 and Caco-2 cells.

RT-PCR of RNA isolated from monolayers of the human colonic epithelial cell lines T84 and Caco-2 demonstrated the presence of mRNA for the two cloned Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2, but not for the cloned Na+-dependent concentrative nucleoside transporters, CNT1 and CNT2. Uptake of [3H]uridine by cell monolayers in balanced Na+-containing and Na+-free media confirmed the presence of only Na+-independent nucleoside transport mechanisms. This uptake was decreased by 70-75% in the presence of 1 microM nitrobenzylthioinosine, a concentration that completely inhibits ENT1, and was completely blocked by the addition of 10 microM dipyridamole, a concentration that inhibits both ENT1 and ENT2. These findings indicate the presence in T84 and Caco-2 cells of two functional Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2.     

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.     

Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis

Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.     

Interleukin-10 and nerve growth factor have reciprocal upregulatory effects on intestinal epithelial cells

The intestinal mucosa is in a constant state of controlled inflammation, but the processes whereby this occurs are poorly understood. The aims of this study were to look at the role of IL-10 and nerve growth factor (NGF) in intestinal epithelial cell regulation. The human colon epithelial cell lines T84, HT-29, and CACO-2 were used. RT-PCR, flow cytometry analysis, and immunohistochemistry were applied to measure the cytokine changes in epithelial cells induced by recombinant cholera toxin and its B subunit, IL-10, and NGF. Cholera toxin B subunit caused selective dose-dependent increased mRNA for IL-10 in T84 cells and the protein in T84, HT-29, and CACO-2 cells. IL-10 dose dependently selectively increased NGF mRNA in T84 cells and intracellular protein synthesis in all three epithelial cell lines. The effect of NGF was reciprocal, selective, and dose dependent because it increased mRNA for IL-10 and IL-10 synthesis. Our results suggest that the epithelium may actively participate in downregulation through innate mechanisms involving IL-10 and NGF. The reciprocal interaction suggests for the first time that NGF may be involved in local downregulation by mucosal epithelium and thus may play a potent protective role in response to injury, by prevention of undue inflammation.     

Invasion of human epithelial cells by Campylobacter upsaliensis

Few data exist on the interaction of Campylobacter upsaliensis with host cells, and the potential for this emerging enteropathogen to invade epithelial cells has not been explored. We have characterized the ability of C. upsaliensis to invade both cultured epithelial cell lines and primary human small intestinal cells. Epithelial cell lines of intestinal origin appeared to be more susceptible to invasion than non-intestinal-derived cells. Of three bacterial isolates studied, a human clinical isolate, CU1887, entered cells most efficiently. Although there was a trend towards more efficient invasion of Caco-2 cells by C. upsaliensis CU1887 at lower initial inocula, actual numbers of intracellular organisms increased with increasing multiplicity of infection and with prolonged incubation period. Confocal microscopy revealed C. upsaliensis within primary human small intestinal cells. Both Caco-2 and primary cells in non-confluent areas of the infected monolayers were substantially more susceptible to infection than confluent cells. The specific cytoskeletal inhibitors cytochalasin B, cytochalasin D and vinblastine attenuated invasion of Caco-2 cells in a concentration-dependent manner, providing evidence for both microtubule- and microfilament-dependent uptake of C. upsaliensis. Electron microscopy revealed the presence of organisms within Caco-2 cell cytoplasmic vacuoles. C. upsaliensis is capable of invading epithelial cells and appears to interact with host cell cytoskeletal structures in order to gain entry to the intracellular environment. Entry into cultured primary intestinal cells ex vivo provides strong support for the role of host cell invasion during human enteric C. upsaliensis infection.     

Fractalkine is an epithelial and endothelial cell-derived chemoattractant for intraepithelial lymphocytes in the small intestinal mucosa

Fractalkine is a unique chemokine that combines properties of both chemoattractants and adhesion molecules. Fractalkine mRNA expression has been observed in the intestine. However, the role of fractalkine in the healthy intestine and during inflammatory mucosal responses is not known. Studies were undertaken to determine the expression and function of fractalkine and the fractalkine receptor CX3CR1 in the human small intestinal mucosa. We identified intestinal epithelial cells as a novel source of fractalkine. The basal expression of fractalkine mRNA and protein in the intestinal epithelial cell line T-84 was under the control of the inflammatory mediator IL-1beta. Fractalkine was shed from intestinal epithelial cell surface upon stimulation with IL-1beta. Fractalkine localized with caveolin-1 in detergent-insoluble glycolipid-enriched membrane microdomains in T-84 cells. Cellular distribution of fractalkine was regulated during polarization of T-84 cells. A subpopulation of isolated human intestinal intraepithelial lymphocytes expressed the fractalkine receptor CX3CR1 and migrated specifically along fractalkine gradients after activation with IL-2. Immunohistochemistry demonstrated fractalkine expression in intestinal epithelial cells and endothelial cells in normal small intestine and in active Crohn's disease mucosa. Furthermore, fractalkine mRNA expression was significantly up-regulated in the intestine during active Crohn's disease. This study demonstrates that fractalkine-CX3CR1-mediated mechanism may direct lymphocyte chemoattraction and adhesion within the healthy and diseased human small intestinal mucosa.