A mucosal intranet: intestinal epithelial cells down-regulate intraepithelial, but not peripheral, T lymphocytes

Epithelial cells and lymphocytes, including gammadelta and alphabeta T cells, in the gastrointestinal tract epithelium represent a major host defense intranet that is incompletely understood. Cell-to-cell interactions between intraepithelial lymphocytes (IELs) and intestinal epithelial cells (IECs) comprise this intranet, and we have assessed the role of IECs in the regulation of gammadelta and alphabeta T cell responses. When highly purified CD3+ IEL T cells were stimulated via the TCR-CD3 complex, high proliferative responses and cytokine synthesis were induced. However, the addition of viable IECs or purified IEC membranes (mIEC) down-regulated T cell proliferative and cytokine responses. Further, the inhibitory effect of mIEC was not restored by antibodies to TGF-beta, CD1d, E-cadherin, or MHC class I or II. This inhibitory effect was noted for both gammadelta and alphabeta T cell subsets from IELs, and mRNA levels were reduced for both Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-5) cytokines in gammadelta and alphabeta IELs. In contrast, a purified membrane fraction obtained from thymocytes did not inhibit IEL proliferative responses. Further, mIEC did not inhibit splenic alphabeta T cell proliferative responses. These findings show that cell-to-cell interactions between intraepithelial gammadelta and alphabeta T cells and IECs occur via cell surface molecules, suggesting an intranet to prevent potential inflammatory responses at the intestinal mucosal surface.     

Differential effects of mucosal pH on human (Caco-2) intestinal epithelial cell motility, proliferation, and differentiation

Mucosal pH abnormalities are associated with anastomotic dehiscence, ischemia, and malignancy. We postulated that intraluminal pH influences intestinal epithelial motility, proliferation, and differentiation and studied extracellular pHo (7.0-8.5) effects on human (Caco-2) intestinal epithelial motility, proliferation, and differentiation. Mucosal healing was modeled by sheet migration and differentiation by alkaline phosphatase and dipeptidyl dipeptidase specific activity. In parallel differentiation and motility studies, we inhibited proliferation with mitomycin to dissociate indirect mitogenic effects. Intracellular pHi was quantitated using BCECF/AM at varying extracellular pHo and in migrating cells. Motility was maximal at pHo 7.6 and proliferation at 7.2. Each decreased with acidity and alkalinity. By contrast, brush border enzyme activity was lowest at pHo 7.0 and highest at pHo 8.5. pHi was highest at pHo 8.5. Migrating cell pHi was higher than static cell pHi. Thus, extracellular pHo deviations perturb Caco-2 pHi homeostasis and motility. Alkalinity promotes differentiation while acidity induces proliferation and limits differentiation.     

Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

The enzyme nitric oxide synthase catalyzes the oxidation of the amino acid L-arginine to L-citrulline and nitric oxide in an NADPH-dependent reaction. Nitric oxide plays a critical role in signal transduction pathways in the cardiovascular and nervous systems and is a key component of the cytostatic/cytotoxic function of the immune system. Characterization of nitric oxide synthase substrates and cofactors has outlined the broad details of the overall reaction and suggested possibilities for chemical steps in the reaction; however, the molecular details of the reaction mechanism are still poorly understood. Recent evidence suggests a role for the reduced bound pterin in the first step of the reaction--the hydroxylation of L-arginine.     

Factors affecting Caco-2 intestinal epithelial cell interleukin-6 secretion

Intestinal epithelial cells (IEC) have previously been shown to produce several cytokines including interleukin-6 (IL-6). However, many factors which may regulate IL-6 secretion by human IEC still remain a mystery due in part to the lack of appropriate model cell lines and the difficulty of culturing human IEC over long periods of time. We have determined that the human colonic carcinoma cell line Caco-2 is capable of secreting IL-6 when stimulated by the inflammatory cytokines IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and stimulation of these cells with IL-1beta plus TNF-alpha induced a synergistic enhancement of IL-6 secretion. The inflammatory cytokine-induced enhancement in IL-6 secretion was greatest when the cells were cultured in a 10% CO2 atmosphere as compared to cells grown in 5% CO2, suggesting that environmental CO2 levels may affect IEC cytokine secretion. Finally, long-term culture of the Caco-2 cells to induce cellular differentiation had no effect on the capacity of these cells to produce IL-6, indicating that the regulation of IL-6 secretion was not affected by differentiation. Taken together, these studies provide important information on the factors which regulate IL-6 secretion by human IEC as they may contribute to the cytokine network during a mucosal inflammation. The results also suggest that the Caco-2 cell line is an appropriate model for further studies on the regulation of cytokine secretion by human IEC.     

Crohn's disease and ulcerative colitis mucosal T cells are stimulated by intestinal epithelial cells: implications for immunosuppressive therapy

BACKGROUND: Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases, and their pathogenesis is attributed, in part, to alterations of the mucosal immune system. This study was designed to define the possible contribution of epithelial cells to the activation of lamina propria T lymphocytes (LPTs) in CD and UC. METHODS: LPTs isolated from CD, UC, and control surgical specimens were cocultured with freshly isolated allogeneic or autologous epithelial cells or epithelial cell lines. Resulting T-cell proliferation was evaluated by tritiated thymidine incorporation on day 5. RESULTS: When intestinal epithelial cells were used to stimulate mucosal T-cell proliferation, CD and UC LPTs were less responsive than control LPTs (p < 0.05 and p < 0.03, respectively). This difference between inflamed and control T cells was consistently observed by using a variety of different intestinal epithelial cell types. CONCLUSIONS: CD and UC mucosal T cells are hyporesponsive to activation by intestinal epithelial cells when compared with control LPTs. Elucidating the mechanism underlying the differential activation of CD and UC LPTs may help to better understand the immunopathogenesis of these conditions.     

Gap junctional communication between murine macrophages and intestinal epithelial cell lines

In intestinal inflammation, inflammatory cells infiltrate the submucosa and are found juxtaposed to intestinal epithelial cell (IEC) basolateral membranes and may directly regulate IEC function. In this study we determined whether macrophage (M phi), P388D1 and J774A.1, are coupled by gap junctions to IEC lines, Mode-K and IEC6. Using flow cytometric analysis, we show bi-directional transfer of the fluorescent dye, calcein (700 Da) between IEC and M phi resulting in a 3.5-20-fold increase in recipient cell fluorescence. Homocellular and heterocellular dye transfer between M phi and/or IEC was detected in cocultures of P388D1, J774A.1, Mode-K, IEC6 and CMT93. However, transfer between P388D1 and Mode-K was asymmetrical in that transfer from P388D1 to Mode-K was always more efficient than transfer from Mode-K to P388D1. Dye transfer was strictly dependent on IEC-M phi adhesion which in turn was dependent on the polarity of IEC adhesion molecule expression. Both calcein dye transfer and adhesion were inhibited by the addition of heptanol to cocultures. Furthermore we demonstrate both IEC homocellular, and M phi-IEC heterocellular propagation of calcium waves in response to mechanical stimulation, typical of gap junctional communication. Finally, areas of close membrane apposition were seen in electron micrographs of IEC-M phi cocultures, suggestive of gap junction formation. These data indicate that IEC and M phi are coupled by gap junctions suggesting that gap junctional communication may provide a means by which inflammatory cells might regulate IEC function.     

Active secretion of the fluoroquinolone ciprofloxacin by human intestinal epithelial Caco-2 cell layers

The bidirectional transepithelial fluxes of ciprofloxacin, an antibacterial fluoroquinolone, across the human intestinal epithelial Caco-2 cell-line show marked asymmetry. Basal-to-apical flux of ciprofloxacin (10 microM) exceeds apical-to-basal flux indicating net secretion. Net ciprofloxacin secretion is abolished by azide/2-deoxy-D-glucose treatment, displays saturation kinetics (Km = 0.89 +/- 0.23 mM, Vmax 44.3 +/- 4.9 nmol cm-2.h) and competition by other fluoroquinolones. A specific, active secretion in Caco-2 epithelia may explain the transintestinal elimination of ciprofloxacin observed in pharmacokinetic studies in man.     

Iron absorption in the rat: the search for possible intestinal mucosal carriers

The biological relevance of four iron-containing fractions previously detected in rat intestinal mucosal cells has been studied. The distribution of iron in these fractions obtained by chromatography on Sepharose 6B has been examined after in vivo and in vitro incubation of mucosal cells with 59Ce. In addition, the effects of phenobarbitone, cycloheximide, iron-deficiency and iron-loading on the uptake and distrubution of iron within the four mucosal cell fractions was studied. The iron in fraction I was mostly bound to intracellular membrane particles. Fraction II was shown to be ferritin. Fraction III contained some transferrin and also a protein of molecular weight similar to transferrin but which was not precipitable by antitransferrin antiserum. Quantited with the results of 'chaser' experiments suggested that, in addition to ferritin, at least two of the fractions (I and III) were involved in the process of iron absorption by the mucosal cell.     

Molecular and cellular bases of intestinal epithelial cell invasion by Shigella flexneri

A key step in the pathogenesis of shigellosis is the capacity of the causative bacteria, shigellae, to invade colonic and rectal epithelial cells in humans. This invasive process encompasses several steps: entry into epithelial cells by induction of a macropinocytic event caused by secreted Ipa proteins. the bacterium then escapes from the vacuole and reaches the cytoplasmic compartment in which it divides rapidly and becomes motile via the expression of a surface protein, IcsA, whose polar localization achieves directed polymerization of actin filaments that push the bacterial body forward. Bacteria then engage the inner face of the cellular membrane in the junctional area and form protrusions allowing their passage into the adjacent cell. Lysis of the double membrane eventually allows access to the cytoplasmic compartment of the adjacent cell, thus providing the bacterium with a very efficient mechanism of epithelial colonization.     

The endocytosis of transferrin by rat intestinal epithelial cells

BACKGROUND/AIMS: The transferrin receptor is a prominent protein on the basal and lateral membranes of intestinal epithelial cells, yet little is known of the function of the receptor in the intestine. The aim of the present study was to determine whether intestinal transferrin receptors were capable of facilitating transferrin internalization. METHODS: Using the rat as an experimental model, the uptake of radiolabeled transferrin by cells isolated from different regions along the crypt-villus axis of the proximal small intestine was studied. RESULTS: An intestinal epithelial cell fraction highly enriched in crypt cells bound most radiolabeled transferrin. Cells in this fraction were able to internalize transferrin and recycle it back to the cell surface. A high affinity, saturable pathway of transferrin uptake by these cells predominated at transferrin concentrations below 0.3 mumol/L, whereas at higher concentrations, most uptake was via a nonsaturable process. Intravenously injected radiolabeled transferrin could be detected within intestinal crypt cells, indicating that these cells are able to internalize transferrin in vivo. CONCLUSIONS: These data suggest that intestinal crypt cells have an active transferrin/transferrin receptor system. Transferrin may play an important role in iron delivery to and/or as a growth factor for the rapidly proliferating intestinal epithelium.     

The effect of apotransferrin on iron release from Caco-2 cells, an intestinal epithelial cell line

The Caco-2 cell line grown in bicameral chambers was used to study the effect of transferrin in the basal chamber on the transepithelial transport of iron. We have shown that when iron was offered as 59Fe on the apical surface of the Caco-2 cells, transport of 59Fe into the basal chamber was stimulated by 50 micromol/L apotransferrin. Here, we examined the effect on 59Fe transport of lower concentrations of apotransferrin, as well as the effects on transport of ovo-, cobalt-, and ferri-transferrin and of iron chelators with an affinity for iron greater than that of transferrin. The stimulation of 59Fe transport was more sensitive to the presence of apotransferrin with a Km of 0.078 +/- 0.008 micromol/L compared with ferri-transferrin with a Km of 1.24 +/- 0.39 micromol/L (P < .006). 59Fe transport was less sensitive to diethylenetriaminopenta-acetic acid (DTPA) than apotransferrin with Kms of 1.52 +/- 0.70. The chelator nitrilotriacetic acid (NTA) exhibited no stimulation of 59Fe transport. Analysis of laser scanning confocal micrographs showed that apotransferrin labeled with Texas Red is internalized by Caco-2 cells from the basal side and localizes in distinct vesicles above the nucleus. The sensitivity of apotransferrin in stimulating Fe transport suggests a unique interaction of apotransferrin with the basal surface of the intestinal epithelium.     

Modulation of human CACO-2 intestinal epithelial cell phenotype by protein kinase C inhibitors

Protein kinase C (PKC) isoforms are altered in colon tumors and upon exposure of intestinal mucosa to nutrients. We evaluated the effects of the PKC inhibitors staurosporine and calphostin C on human Caco-2 intestinal epithelial proliferation, motility, and differentiation. Motility was quantitated by monolayer expansion and the brush border enzymes dipeptidyl dipeptidase (DPDD) and alkaline phosphatase (AP) by synthetic substrate digestion. Staurosporine (0.03-1.0 ng/ml) and calphostin C (10(-12) M-10(-4) M) dose-dependently inhibited monolayer expansion and AP but stimulated DPDD. Proliferation was also inhibited but the effects of each inhibitor on motility, AP, and DPDD were preserved after mitomycin C proliferative blockade, suggesting that these effects were proliferation-independent. PKC inhibitors independently inhibit motility, AP and proliferation in human intestinal Caco-2 epithelial cells, but selectively stimulate the small intestinal differentiation marker DPDD. PKC may regulate small intestinal epithelial differentiation.     

Yersinia enterocolitica-induced interleukin-8 secretion by human intestinal epithelial cells depends on cell differentiation

In response to bacterial entry epithelial cells up-regulate expression and secretion of various proinflammatory cytokines, including interleukin-8 (IL-8). We studied Yersinia enterocolitica O:8-induced IL-8 secretion by intestinal epithelial cells as a function of cell differentiation. For this purpose, human T84 intestinal epithelial cells were grown on permeable supports, which led to the formation of tight monolayers of polarized intestinal epithelial cells. To analyze IL-8 secretion as a function of cell differentiation, T84 monolayers were infected from the apical or basolateral side at different stages of differentiation. Both virulent (plasmid-carrying) and nonvirulent (plasmid-cured) Y. enterocolitica strains invaded nondifferentiated T84 cells from the apical side. Yersinia invasion into T84 cells was followed by secretion of IL-8. After polarized differentiation of T84 cells Y. enterocolitica was no longer able to invade from the apical side or to induce IL-8 secretion by T84 cells. However, Y. enterocolitica invaded and induced IL-8 secretion by polarized T84 cells after infection from the basolateral side. Basolateral invasion required the presence of the Yersinia invasion locus, inv, suggesting beta1 integrin-mediated cell invasion. After basolateral infection, Yersinia-induced IL-8 secretion was not strictly dependent on cell invasion. Thus, although the plasmid-carrying Y. enterocolitica strain did not significantly invade T84 cells, it induced significant IL-8 secretion. Taken together, these data show that Yersinia-triggered IL-8 secretion by intestinal epithelial cells depends on cell differentiation and might be induced by invasion as well as by basolateral adhesion, suggesting that invasion is not essential for triggering IL-8 production. Whether IL-8 secretion is involved in the pathogenesis of Yersinia-induced abscess formation in Peyer's patch tissue remains to be shown.     

Toxic epidermal necrolysis with severe gastrointestinal mucosal cell death: a patient who excreted long tubes of dead intestinal epithelium

TEN is a severe inflammatory disease which is characterized by generalized epithelial destruction. The epidermis is the most common target of TEN, however, any epithelium can be involved. We report a toxic epidermal necrolysis (TEN) patient who excreted long tubes of dead intestinal epithelium. Epidermal keratinocytes and intestinal epithelium were found to undergo extensive apoptosis by TUNEL method. Drugs were speculated as the causative agents for this case, the causative drug has not been identified. In contrast to marked improvement of cutaneous manifestation and hepatic function by methyl prednisolone pulse therapy, the gastrointestinal symptoms did not respond to therapies, and the patient died by heart failure. Present case suggested a pathogenetic mechanism targeting antigens commonly expressed on the gastrointestinal epithelium and epidermis.     

The neuronal stem cell of the olfactory epithelium

Reviews and evaluates psychosocial treatments for attention deficit hyperactivity disorder (ADHD) in children and adolescents according to Task Force Criteria (Lonigan, Elbert, & Johnson, this issue). It is concluded that behavioral parent training and behavioral interventions in the classroom meet criteria for well-established treatments. Cognitive interventions do not meet criteria for well-established or probably efficacious treatments. Issues regarding the evaluative process are discussed and future directions for psychosocial treatment for ADHD are outlined.     

Adherence to and penetration of human intestinal Caco-2 epithelial cell monolayers by Pseudomonas aeruginosa

Clinical isolates of Pseudomonas aeruginosa from blood adhered to and penetrated intestinal Caco-2 cell monolayers to a greater degree than did isolates from sputum, with a concomitant drastic decrease in transepithelial electrical resistance. PAO-PR1, an avirulent exotoxin A mutant of PAO1, did not cause a decrease in the resistance. The Caco-2 monolayer system may be useful for the evaluation of certain P. aeruginosa virulence factor activities.     

Age-dependent intestinal lymphoproliferative disorder due to stem cell factor receptor deficiency: parameters in small and large intestine

Signaling through c-Kit/stem cell factor (SCF) is crucial for normal development of erythroid and myeloid hematopoietic precursors and of melanocytes and germ cells. While peripheral lymphoid populations of W/Wv and SI/SId mice appear normal, we demonstrated that the intraepithelial lymphocyte (IEL) populations of small (SI) and large (LI) intestine were significantly affected. IEL populations of young W/Wv animals were indistinguishable from those of their control littermates, but an age-dependent decrease in SI and LI TCRgamma delta IEL occurred in c-Kit mutant mice. In SI, but not in LI, this diminution was accompanied by gross expansion of TCRalpha beta IEL that resulted in significantly increased IEL:epithelial cell ratios in c-Kit mutant mice. Bromodeoxyuridine labeling studies revealed that the increase in cell numbers was due to lymphoproliferation that occurred in situ. Interestingly, TCRgamma delta IEL expressed cell surface c-Kit, while the expanding population of TCRalpha beta IEL did not. Analysis of radiation bone marrow chimeras demonstrated that the dysregulation required either disruption of stromal cell SCF or IEL c-Kit and showed that the effect on IEL or their precursors was not due to other changes in the intestinal microenvironment. Lamina propria T cell populations in these mice were unaffected, reinforcing the idea that the developmental requirements of these gut-resident lymphocyte populations are distinct. Overall, the results demonstrated that the development of intestinal TCRgamma delta IEL, regardless of location, shares common requirements for SCF, while SI and LI TCRalpha beta IEL may develop along distinct pathways. Possible mechanisms for the loss of proliferative regulation in gut T cells in c-Kit/SCF deficiency are discussed.