Biomedical subjects

S P Colgan

Publications and source records attributed to S P Colgan.

At least 55 records · Page 3Linked to original sources

PubMed1996

IFN-gamma modulates CD1d surface expression on intestinal epithelia.

In vivo, epithelial cells that line the intestine are intimately associated with lymphocytes, termed intestinal intraepithelial lymphocytes (iIEL). A putative ligand for iIEL on intestinal epithelial cells is CD1d, and recent studies demonstrate a surface form of this molecule exists on intestinal epithelia. At present, it is not known whether CD1d expression is regulated by cytokines in the intestinal microenvironment. Thus we examined the impact of relevant cytokines on CD1d at the level of mRNA and cell surface expression. Using a sensitive whole cell enzyme-linked immunosorbent assay, we assessed the impact of relevant cytokines on CD1d expression on intestinal epithelial cell lines. We were readily able to detect CD1d on the surface of T84 cells, a cryptlike intestinal epithelial cell line. Epithelial cell exposure to human recombinant interferon-gamma (IFN-gamma) resulted in increased CD1d expression in a dose- and time-dependent manner. Polymerase chain reaction amplification of CD1d cDNA revealed a time-dependent induction after exposure to IFN-gamma. This IFN-gamma effect on CD1d expression was cytokine specific and was evident with epithelial cell lines other than T84, including Caco-2 and HT-29 cells. Finally, we were not able to detect significant surface expression of CD1a, CD1b, or CD1c on intestinal epithelial cell lines in the presence or absence of relevant cytokines. These results indicate that CD1d cell surface protein and cellular mRNA, like other major histocompatibility complex-related molecules, is cytokine regulated in intestinal epithelial cell lines.

Antigens, CD1↗

PubMed1996

Intestinal epithelial cytoskeleton selectively constrains lumen-to-tissue migration of neutrophils.

Migration of neutrophils (polymorphonuclear leukocytes; PMN) across polarized epithelia is asymmetrical: basolateral-to-apical (physiologically directed) migration is far more efficient than migration in the reverse direction, suggesting the presence of luminal retention signal(s). Following pilot observations, we used polarized intestinal epithelial monolayers (T84) to examine whether asymmetrical constraint of migration afforded by the epithelial cytoskeleton might underlie such retention signals. Rearrangement of epithelial cortical F-actin accompanied PMN transepithelial migration (in either direction) and was prevented by preloading monolayers with the F-actin stabilizing agent phallacidin. Although phallacidin preloading did not influence physiologically directed PMN transepithelial migration, such treatment greatly enhanced migration in the reverse direction (i.e., effective loss of luminal retention signal). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) preloading also prevented epithelial cortical actin rearrangements and selectively resulted in loss of luminal retention signal(s). BAPTA preloading did not influence resistance or forskolin-induced Cl- secretion, and phallacidin preloading did not influence resistance or carbachol-induced Cl- secretion, suggesting that barrier function and surface polarity were maintained under these conditions. These and supplementary data suggest that epithelial actin (but not microtubule) cytoskeletal reordering asymmetrically influences PMN migration and underlies, at least in part, the observed signal that biases for retention of PMN in the luminal space.

Actins↗

PubMed1996

Hypoxic stress alone does not modulate endothelial surface expression of bovine E-selectin and intercellular adhesion molecule-1 (ICAM-1).

UNLABELLED: Hypoxemia is a common event in many vascular diseases, especially vascular ischemia. Since endothelial cells of blood vessels are exposed to conditions within the vascular space and leucocytes play a key role in ischemia/reperfusion injury, we hypothesized that endothelial exposure to hypoxia may regulate expression of surface proteins important in leucocyte-endothelial interactions, such as E-selectin and intercellular adhesion molecule (ICAM-1). In this study, we used isolated bovine aortic endothelial monolayers to examine endothelial surface alterations of E-selectin and ICAM-1 induced by tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) and hypoxia using a whole cell enzyme-linked immunosorbent assay (ELISA). Bovine endothelial exposure to TNF-alpha (50 ng/mL) induced a time dependent increase (range 0-24h) in specific E-selection surface expression. Endothelial exposure to hypoxia alone (pO2 approximately 3 mmHg, range 0-24 h), however, failed to elicit endothelial E-selectin expression. Endothelial exposure to LPS brought about a dose- and time-dependent (range 0.5 ng/mL and 2-8 h) increase in specific ICAM-1 surface expression (max. 3.5 +/- 0.15-fold increase over no cytokine control at 10 ng/mL, 4 h). Hypoxia (pO2 approximately 3 mmHg, 8h), however, did not induce ICAM-1 surface expression over normoxia levels. IN CONCLUSION: i) bovine endothelial E-selectin and ICAM-1 surface expression are regulated molecules, ii) hypoxia, per se, does not regulate surface expression of either E-selectin or ICAM-1. These results suggest that hypoxic endothelia may require additional external signals for generation of adaptive inflammatory responses.

Animals↗

PubMed1996

Expression and polarization of intercellular adhesion molecule-1 on human intestinal epithelia: consequences for CD11b/CD18-mediated interactions with neutrophils.

BACKGROUND: Epithelial dysfunction and patient symptoms in inflammatory intestinal diseases such as ulcerative colitis and Crohn's disease correlate with migration of neutrophils (PMN) across the intestinal epithelium. In vitro modeling of PMN transepithelial migration has revealed distinct differences from transendothelial migration. By using polarized monolayers of human intestinal epithelia (T84), PMN transepithelial migration has been shown to be dependent on the leukocyte integrin CD11b/CD18 (Mac-1), but not on CD11a/CD18 (LFA-1). Since intercellular adhesion molecule-I (ICAM-1) is an important endothelial counterreceptor for these integrins, its expression in intestinal epithelia and role in PMN-intestinal epithelial interactions was investigated. MATERIALS AND METHODS: A panel of antibodies against different domains of ICAM-1, polarized monolayers of human intestinal epithelia (T84), and natural human colonic epithelia were used to examine the polarity of epithelial ICAM-1 surface expression and the functional role of ICAM-1 in neutrophil-intestinal epithelial adhesive interactions. RESULTS: While no surface expression of ICAM-1 was detected on unstimulated T84 cells, interferon-gamma (IFN gamma) elicited a marked expression of ICAM-1 that selectively polarized to the apical epithelial membrane. Similarly, apically restricted surface expression of ICAM-1 was detected in natural human colonic epithelium only in association with active inflammation. With or without IFN gamma pre-exposure, physiologically directed (basolateral-to-apical) transepithelial migration of PMN was unaffected by blocking monoclonal antibodies (mAbs) to ICAM-1. In contrast, PMN migration across IFN gamma-stimulated monolayers in the reverse (apical-to-basolateral) direction was inhibited by anti-ICAM-1 antibodies. Adhesion studies revealed that T84 cells adhered selectively to purified CD11b/CD18 and such adherence, with or without IFN gamma pre-exposure, was unaffected by ICAM-1 mAb. Similarly, freshly isolated epithelial cells from inflamed human intestine bound to CD11b/CD18 in an ICAM-1-independent fashion. CONCLUSIONS: These data indicate that ICAM-1 is strictly polarized in intestinal epithelia and does not represent a counterreceptor for neutrophil CD11b/CD18 during physiologically directed transmigration, but may facilitate apical membrane-PMN interactions after the arrival of PMN in the intestinal lumen.

Antibodies, Monoclonal↗

PubMed1995

Design of lipoxin A4 stable analogs that block transmigration and adhesion of human neutrophils.

Lipoxins (LX) are bioactive eicosanoids that carry a tetraene structure and serve as regulators of inflammation, in part by inhibiting neutrophil migration and adhesion. Lipoxin A4 is rapidly regulated by conversion to inactive LX metabolites via local metabolism that involves dehydrogenation as the predominant route. Here, several LXA4 analogs were designed that resisted rapid conversion by both differentiated HL-60 cells and recombinant 15-hydroxyprostaglandin dehydrogenase, systems where native LXA4 is degraded within minutes. The rank order of conversion by recombinant dehydrogenase was LXA4 methyl ester > PGE2 approximately PGE2 methyl ester > LXA4 >>> the novel LXA4 analogs. In addition, 15(R/S)-methyl-LXA4, 15-cyclohexyl-LXA4, and 16-phenoxy-LXA4 proved to retain LXA4 bioactivity and inhibited neutrophil transmigration across polarized epithelial cell monolayers as well as adhesion to vascular endothelial cells. These results indicate that LXA4 analogs can be designed using these criteria to resist rapid transformation and to retain biological actions of native LXA4. Moreover, the results suggest that LXA4 stable analogs can be useful tools both in vitro and in vivo to evaluate LXA4 actions and therapeutic potential.

Cell Adhesion↗

PubMed1995

Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia.

The rho family of GTP-binding proteins regulates actin filament organization. In unpolarized mammalian cells, rho proteins regulate the assembly of actin-containing stress fibers at the cell-matrix interface. Polarized epithelial cells, in contrast, are tall and cylindrical with well developed intercellular tight junctions that permit them to behave as biologic barriers. We report that rho regulates filamentous actin organization preferentially in the apical pole of polarized intestinal epithelial cells and, in so doing, influences the organization and permeability of the associated apical tight junctions. Thus, barrier function, which is an essential characteristic of columnar epithelia, is regulated by rho.

ADP Ribose Transferases↗

PubMed1995

Receptors involved in carbohydrate binding modulate intestinal epithelial-neutrophil interactions.

Neutrophil (polymorphonuclear neutrophil) migration across epithelial barriers is a common morphologic feature of many diseases. Previous studies show that PMN-epithelial interactions are dependent on the PMN beta 2-integrin CD11b/18; however, nothing is known about surface carbohydrates and PMN-epithelial interactions. Here we investigate the role of carbohydrates on PMN-epithelial interactions using PMN and cultured monolayers of the intestinal epithelial cell line T84. Addition of the carbohydrates mannose 6-phosphate (Man-6-P) and glucose 6-phosphate (Glu-6-P), but not fructose 1-phosphate (Fru-1-P) inhibited transmigration by > or = 70%. Likewise, more complex carbohydrates, such as fucoidin and the Man-6-P-rich polysaccharide PPME selectively inhibited PMN transepithelial migration. These carbohydrates were found to be inhibitory in the apical-to-basolateral direction as well as the basolateral-to-apical direction, indicating a lack of polarity. This panel of related carbohydrates, however, was not effective in modulating short-term adhesion of PMN to epithelial monolayers, indicating that carbohydrate ligands may modulate different steps in the transmigration cascade. Finally, addition of functionally inhibitory monoclonal antibodies specific for the selectins (CD62E, CD62L, and CD62P) revealed no observable effect on PMN transmigration. These studies suggest that cell surface carbohydrates may play a role in inflammatory processes of the intestine.

Carbohydrate Metabolism↗

PubMed1995

Human eosinophils migrate across an intestinal epithelium in response to platelet-activating factor.

BACKGROUND/AIMS: Transmigration of eosinophils across intestinal epithelia occurs in a variety of mucosal inflammatory disorders and results in the formation of crypt abscesses containing eosinophils. However, the conditions required to drive transepithelial migration of eosinophils are not understood. METHODS: This study investigated eosinophil migration across intestinal epithelia using purified eosinophils and confluent monolayers of the human intestinal epithelial cell line T84. RESULTS: Unactivated eosinophils (i.e., no granulocyte/macrophage colony-stimulating factor [GM-CSF] preexposure) did not migrate across T84 monolayers in the presence of transepithelial gradients of C5a, n-formyl-methionyl-leucyl-phenylalanine (fMLP), or platelet-activating factor (PAF). In contrast, activation of eosinophils by coincubation or pretreatment with GM-CSF enabled transepithelial migration in response to PAF but not to C5a or fMLP gradients in a time- and dose-dependent fashion. Specificity was confirmed by both the PAF receptor antagonist WEB-2086 and the PAF enantiomer 2-lyso-beta-acetyl-gamma-O-hexadecyl. Finally, addition of functionally inhibitory monoclonal antibodies to CD11b but not CD11a, very late activation antigen (VLA-4), or intracellular adhesion molecule 1 inhibited eosinophil migration. CONCLUSIONS: These studies establish that physiologically directed migration of eosinophils across model epithelia occurs but that this process is governed by the state of eosinophil activation, the specific chemotactic gradient imposed, and the availability of specific surface integrins to participate in putative eosinophil-epithelial adhesion steps.

Adult↗

PubMed1995

Intestinal epithelia (T84) possess basolateral ligands for CD11b/CD18-mediated neutrophil adherence.

The process of active intestinal inflammation can be modeled by the migration of neutrophils [polymorphonuclear leukocytes (PMN)] across monolayers of the human intestinal cell line T84. We now evaluate the polarity of early adhesive events occurring between human PMN and ligands on intact T84 monolayers. Transient Ca2+ depletion (tr-Ca-depl) of confluent monolayers grown on permeable supports (2 mM EDTA, 12 min, 37 degrees C) resulted in disruption of tight junctions separating apical and basolateral domains (resistance fell from 973 +/- 35 to 57.4 +/- 3.8 omega.cm2 after 12 min). The columnar T84 phenotype was not grossly altered by tr-Ca-depl. Access to basolateral membrane proteins from the apical surface after tr-Ca-depl was confirmed by enzyme-linked immunosorbant assay and immunofluorescence, using antibodies to the basolateral proteins E-Cadherin and beta 1-integrin. The effects of tr-Ca-depl on early events in PMN adhesion were next assessed. Neutrophils (2 x 10(6)) were applied to the apical surface of monolayers followed by stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (100 nM, 5 min, 37 degrees C) and quantitation. In comparison with control monolayers, tr-Ca-depl increased subsequent PMN adhesion (1.7 +/- 0.24 vs. 3.1 +/- 0.28 x 10(4) adherent PMN/monolayer; P < 0.005), which was inhibited 80% by anti-CD11b/CD18 antibodies. These experiments suggest that PMN adhere to subjunctional ligands on T84 cells in a CD11b/CD18-dependent fashion.

CD18 Antigens↗

PubMed1995

Assessment of inflammatory events in epithelial permeability: a rapid screening method using fluorescein dextrans.

Cultured intestinal epithelial monolayers serve as models for mechanistic studies of intestinal inflammation. One crucial aspect of epithelial function modulated by inflammation is permeability. Indices of permeability typically obtained are transepithelial resistance or more formalized assays using Ussing chambers modified for cultured monolayers. Such Ussing chamber systems are inconvenient for screening assays and their geometry precludes evaluation of important inflammatory responses such as transepithelial migration of neutrophils. Here we use a novel flux assay to investigate the kinetics of fluorescein-labelled dextran (FD) molecules across intestinal epithelial cell lines grown on permeable membrane supports. Our results show that we could consistently demonstrate picomolar flux of FD in a range of sizes (3-70 kD) across T84 epithelial monolayers. Such FD flux was time- and dose-dependent and flux increased exponentially with decreasing transepithelial resistance. Exposure of intestinal epithelia to mucosal-derived lymphocytes or to soluble lymphocyte products (interferon-gamma, interleukin-4 or interleukin-13) increased FD flux in a dose-dependent fashion. Finally, studies of neutrophil transepithelial migration revealed qualitative and quantitative differences in FD flux depending on FD size. We conclude that in vitro transepithelial FD flux may be a useful tool to study aspects of intestinal permeability in health and in disease.

Biological Transport↗

PubMed1994

IL-4 directly modulates function of a model human intestinal epithelium.

Intestinal epithelia are in intimate contact with subepithelial and intraepithelial lymphocytes. When stimulated, mucosal lymphocytes generate inflammatory cytokines such as IL-4 and IFN-gamma. We have shown that IFN-gamma directly regulates epithelial function. It is unknown whether IL-4 might influence epithelial function and, if so, whether such influences are similar to or differ from those exerted by IFN-gamma. In this study, we examine the effect of human IL-4 on barrier function, ion transport, and immune accessory ligand expression on T84 cells, a crypt-like epithelial cell line. Basolateral exposure of epithelial monolayers to IL-4 attenuated epithelial barrier function by greater than 65% in a dose (50% of effective dose = 1 U/ml)- and time (t1/2 = 24 h)-dependent fashion, and was inhibitable by neutralizing anti-IL-4 and anti-IL-4R Ab. Stimulated Cl- secretion, as measured by epithelial short circuit current, was diminished by as much as 70% by IL-4. Epithelial preexposure to IL-4 brought about a greater than twofold increase in beta 2 integrin-dependent neutrophil adhesion to epithelial, but retarded neutrophil migration into and across epithelial monolayers. ELISAs revealed that epithelial exposure to IL-4 had no effect on cell surface expression of MHC class I, MHC class II, or ICAM-1. These results indicate that IL-4, like IFN-gamma, may serve to regulate intestinal epithelial function, but that resulting phenotypes may be cytokine specific. We speculate from these data that activation of the basolateral receptor for IL-4 potentially provides a new strategy for damping the cellular component of active inflammation in the intestine.

Cell Adhesion Molecules↗

PubMed1994

Beta 2-microglobulin-independent MHC class Ib molecule expressed by human intestinal epithelium.

A major histocompatibility complex class Ib protein, CD1d, is expressed by human intestinal epithelial cells (IECs) and is a ligand for CD8+ T cells. CD1d was found to be expressed on the surface of human IECs as a 37-kilodalton protein that was beta 2-microglobulin (beta 2M) independent with no N-linked carbohydrate. Transfection into a beta 2M- cell line confirmed that CD1d could be expressed at the cell surface in the absence of beta 2M. These data indicate that IECs use a specialized pathway for CD1d synthesis and that a beta 2M-independent class Ib protein may be the normal ligand for some intestinal T cells.

Antigens, CD↗

PubMed1994

Interferon-gamma induces a cell surface phenotype switch on T84 intestinal epithelial cells.

Intestinal epithelia are in intimate contact with submucosal and intraepithelial lymphocytes. The concentration of intraepithelial lymphocytes increases during inflammatory processes, and, when stimulated, these cells generate cytokines such as interferon-gamma (IFN-gamma). In this study, we examined the effect of recombinant human IFN-gamma on ion transport events in T84 cells, a crypt epithelial cell line widely used to study electrogenic Cl- secretion, the transport event responsible for mucosal hydration. Epithelial exposure to IFN-gamma brought about a marked attenuation in stimulated Cl- secretion, as measured by generation of short-circuit current (ISC). This IFN-gamma-elicited decrease in the Cl- secretory response was present for a variety of specific agonists, appeared largely due to IFN-gamma interactions with the basolateral surface, and did not result from a defect in second messenger generation. Efflux and uptake studies were utilized to functionally define the individual cell surface transport proteins that participate in Cl- secretion and revealed that, in response to epithelial exposure to IFN-gamma, apical Cl- channels and basolateral Na(+)-K(+)-2Cl- cotransporters, K+ channels, and Na-K-adenosinetriphosphatase were all functionally downregulated. [3H]bumetanide binding assays suggested that surface expression of the cotransporter was diminished by > 70% after IFN-gamma preexposure. Concurrently, surface immunofluorescence studies revealed that epithelial exposure to IFN-gamma brought about the induction of major histocompatibility complex (MHC) class II molecule expression on T84 epithelial monolayers and markedly increased MHC class I surface expression. Finally, neutrophil-epithelial adhesion studies revealed that preexposure of epithelial monolayers to IFN-gamma elicited a beta 2-integrin-dependent induction of neutrophil adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Carrier Proteins↗

PubMed1994

Reconstitution of cultured intestinal epithelial monolayers with a mucosal-derived T lymphocyte cell line. Modulation of epithelial phenotype dependent on lymphocyte-basolateral membrane apposition.

In vivo, epithelial cells which line the intestine are intimately associated with lymphocytes, termed intraepithelial lymphocytes. Previous studies have demonstrated that intraepithelial lymphocytes are present in the uninflamed mucosa, and become especially prominent in various human enteropathies including coeliac disease, tropical sprue, dermatitis herpetiformis, and giardiasis. Using the intestinal crypt cell line T84, and a previously well-defined human mucosa-derived lymphocyte (MDL) line with phenotypic features similar to (but not specific for) intraepithelial lymphocytes, we describe a co-culture model to study the functional sequellae of MDL-T84 cell interactions in vitro. A co-culture method was defined which permitted reconstitution of the paracellular spaces of physiologically confluent epithelial monolayers with MDL. Such co-cultures thus mimicked the correct geometry of intraepithelial lymphocytes-epithelial cell interactions. The presence of physiologically positioned MDL brought about specific and dramatic effects on intestinal epithelial monolayer function. In a dose-dependent fashion, the presence of MDL significantly attenuated barrier function (expressed as a decrease in monolayer resistance), decreased epithelial electrogenic Cl- secretion, and modulated epithelial-neutrophil interactions. Such effects were not reproduced in monolayers similarly reconstituted with inert polystyrene beads equivalent in size to MDL. These MDL-elicited effects on epithelial function specifically required direct MDL apposition to the epithelial basolateral membrane. Furthermore, this specific form of MDL-epithelial basolateral contact released soluble factors which were able to confer the MDL-reconstituted phenotype on virgin epithelial monolayers in the absence of MDL. We have previously shown that many aspects of the MDL converted epithelial phenotype described here can be induced by IFN-gamma. While IFN-gamma, a cytokine produced by many lymphocytes including intraepithelial lymphocytes, was detectable in conditioned supernatants from co-cultures, it existed at concentrations insufficient to fully explain the physiologic effects observed here.

Cells, Cultured↗

PubMed1994

Interactions of neutrophils with epithelial cells: lessons from the intestine.

Active inflammatory processes in organs lined by columnar epithelial cells are often characterized by abundant neutrophil migration. To reach an epithelial surface, neutrophils must leave the vasculature and cross the interstitium where signals may be generated that influence migration and/or epithelial function. Subsequently neutrophils migrate across epithelial linings by impaling intercellular junctions between epithelial cells and, in doing so, diminish the ability of epithelial linings to serve as a barrier. Decreased barrier function brought about by neutrophil migration across epithelia appears to be a reversible process after the removal of the chemotactic stimulus. The molecular events underlying neutrophil-epithelial interactions are at present poorly understood. With recent information regarding neutrophil-endothelial interactions, it has been possible to better understand issues of neutrophil interactions with epithelial cells. In general, there appear to be different rules governing the interaction of neutrophils with endothelial cells as compared with epithelial cells. Neutrophil migration across endothelial monolayers involves a series of at least three steps, each regulated by glycoproteins expressed on the cellular surface. In considering neutrophil-epithelial interactions, the single common denominator is the requirement for surface expression of neutrophil beta 2 integrins, and specifically CD11b/18, as shown by functional inhibition through the use of antibodies to neutrophil beta 2 integrins and the use of neutrophils from patients lacking the beta-chain of this integrin. An understanding of the regulation of neutrophil-epithelial interactions, although in its infancy, appears to involve inflammatory cytokines. Recent in vivo evidence suggests that interferon-gamma production at the level of the intestine may correlate with the regulation of neutrophil-epithelial interactions in vitro by interferon-gamma. Finally, considerable evidence exists that neutrophils may contribute significantly to fluid transport during inflammatory diseases such as secretory diarrhea. When placed in contact with epithelial monolayers, activated neutrophils bring about the transport of ions, with concomitant water loss across epithelial surfaces. This transport process brought about by the presence of neutrophils is generated by a metabolite produced and secreted from neutrophils. This metabolite was originally termed neutrophil-derived secretagogue and has recently been identified as 5'-AMP. The mechanism of 5'-AMP action on epithelial cell ion transport is discussed.

Animals↗

PubMed1993

Activated eosinophils evoke chloride secretion in model intestinal epithelia primarily via regulated release of 5'-AMP.

Eosinophils may be prominent in intestinal diseases including allergic gastroenteritis, inflammatory bowel disease, enteritis associated with hypereosinophilic syndromes (HES), and parasitic diseases. Unlike normal blood eosinophils, those that circulate in HES and those that infiltrate inflamed tissue exhibit an "activated" phenotype. To model intestinal epithelial-eosinophil interactions, we used peripheral blood eosinophils and human crypt-like T84 epithelial cell-line monolayers. Eosinophils from normal, mildly atopic donors, only if activated by PMA or primed with granulocyte-macrophage-CSF for 48 h, as well as eosinophils from HES patients elicited a short circuit current when applied apically to T84 monolayers. This eosinophil-derived bioactivity, which was transferable in cell-free supernatants and in < 1000 m.w. ultrafiltrates, stimulated electrogenic Cl- secretion, as indicated by inhibition with basolateral bumetanide or gluconate substitution and by enhancement of the rate constant for 125I efflux from preloaded T84 cells. This secretagogue activity was blocked in both intact activated eosinophils and in eosinophil-conditioned supernatants, by 8-phenyl-theophylline, indicating involvement of an adenosine receptor. Ion exchange and reversed-phase HPLC analyses demonstrated that eosinophil supernatant ultrafiltrates contained elevated levels of 5'-AMP that was converted to adenosine after incubation with epithelium. Inhibition of epithelial apical membrane ecto-5'-nucleotidase ablated the conversion to adenosine. These studies establish that activated eosinophils elicit Cl- secretion from intestinal epithelial and that 5'-AMP released by eosinophils followed by its conversion to adenosine at the epithelial surface is the basis for this response.

Adenosine Diphosphate↗

PubMed1993

Neutrophil migration across cultured intestinal epithelial monolayers is modulated by epithelial exposure to IFN-gamma in a highly polarized fashion.

Neutrophil, or polymorphonuclear leukocyte (PMN), migration across intestinal epithelial barriers, such as occurs in many disease states, appears to result in modifications of epithelial barrier and ion transport functions (Nash, S., J. Stafford, and J. L. Madara. 1987. J. Clin. Invest. 80:1104-1113; Madara, J. L., C. A. Parkos, S. P. Colgan, R. J. MacLeod, S. Nash, J. B. Matthews, C. Delp, and W. I. Lencer. 1992. J. Clin. Invest. 89:1938-1944). Here we investigate the effects of epithelial exposure to IFN-gamma on PMN migration across cultured monolayers of the human intestinal epithelial cell line T84. Transepithelial migration of PMN was initially assessed in the apical-to-basolateral direction, since previous studies indicate general qualitative similarities between PMN migration in the apical-to-basolateral and in the basolateral-to-apical directions. In the apical-to-basolateral direction, epithelial exposure to IFN-gamma markedly upregulated transepithelial migration of PMN in a dose- and time-dependent fashion as measured by both electrical and myeloperoxidase assays. This IFN-gamma-elicited effect on transmigration was specifically due to a IFN-gamma effect on epithelial cells and was not secondary to IFN-gamma effects on epithelial tight junction permeability. Moreover, this IFN-gamma effect was dependent on epithelial protein synthesis, and involved a pathway in which CD11b/18, but not ICAM-1 or CD11a/18, appeared to play a crucial role in PMN-epithelial adhesion. IFN-gamma also substantially modified PMN transepithelial migration in the natural, basolateral-to-apical direction. The IFN-gamma effect on naturally directed transmigration was also specifically due to an IFN-gamma effect on epithelial cells, showed comparable time and dose dependency to that of oppositely directed migration, was CD11b/18 dependent, and required epithelial protein synthesis. Additionally, however, important qualitative differences existed in how IFN-gamma affected transmigration in the two directions. In contrast to apical-to-basolateral directed migration, IFN-gamma markedly downregulated transepithelial migration of PMN in the natural direction. This downregulation of PMN migration in the natural direction, however, was not due to failure of PMN to move across filters and into monolayers. Indeed, IFN-gamma exposure to epithelia increased the number of PMN which had moved into the basolateral space of the epithelium in naturally directed transmigration. These results represent the first detailed report of influences on PMN transepithelial migration by a cytokine, define conditions under which a qualitative difference in PMN transepithelial migration exists, and suggest that migration of PMN across epithelia in the natural direction may involve multiple steps which can be differentially regulated by cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗

PubMed1993

Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils.

In human intestinal disease induced by Salmonella typhimurium, transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane. In this report, we model those interactions in vitro, using polarized monolayers of the human intestinal epithelial cell, T84, isolated human PMN, and S. typhimurium. We show that Salmonella attachment to T84 cell apical membranes did not alter monolayer integrity as assessed by transepithelial resistance and measurements of ion transport. However, when human neutrophils were subsequently placed on the basolateral surface of monolayers apically colonized by Salmonella, physiologically directed transepithelial PMN migration ensued. In contrast, attachment of a non-pathogenic Escherichia coli strain to the apical membrane of epithelial cells at comparable densities failed to stimulate a directed PMN transepithelial migration. Use of the n-formyl-peptide receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1- phenylalanine (tBOC-MLP) indicated that the Salmonella-induced PMN transepithelial migration response was not attributable to the classical pathway by which bacteria induce directed migration of PMN. Moreover, the PMN transmigration response required Salmonella adhesion to the epithelial apical membrane and subsequent reciprocal protein synthesis in both bacteria and epithelial cells. Among the events stimulated by this interaction was the epithelial synthesis and polarized release of the potent PMN chemotactic peptide interleukin-8 (IL-8). However, IL-8 neutralization, transfer, and induction experiments indicated that this cytokine was not responsible for the elicited PMN transmigration. These data indicate that a novel transcellular pathway exists in which subepithelial PMN respond to lumenal pathogens across a functionally intact epithelium. Based on the known unique characteristics of the intestinal mucosa, we speculate that IL-8 may act in concert with an as yet unidentified transcellular chemotactic factor(s) (TCF) which directs PMN migration across the intestinal epithelium.

Bacterial Adhesion↗