Identification of a human enterocyte lipoxin A4 receptor that is regulated by interleukin (IL)-13 and interferon gamma and inhibits tumor necrosis factor alpha-induced IL-8 release.

Epithelial cells of the alimentary tract play a central role in mucosal immunophysiology. Pathogens and/or agonists that interact with mucosal surfaces often elicit epithelial responses that upregulate inflammation. Therefore, it was of interest to explore potential epithelial targeted antiinflammatory signals. Here we identified and sequenced a human enterocyte lipoxin (LX) A4 [5(S), 6(R),15(S)-trihydroxy-7,9,13-trans-11-cis eicosatetraenoic acid] receptor, and demonstrate that transcription of this receptor was controlled by cytokines, of which lymphocyte-derived interleukin (IL)-13 and interferon gamma were the most potent. When lipoxins and LXA4 stable analogs were evaluated for enterocyte functional as well as immune responses, lipoxins sharply inhibited TNF-alpha-induced IL-8 release but did not alter either barrier function or agonist-stimulated chloride secretion. 15R/S-methyl-LXA4 and 16-phenoxy-LXA4 each attenuated (IC50 approximately 10 nM) IL-8 release. Cyclooxygenase (COX) II is emerging as an important component in wound healing and proliferation in intestinal epithelia and when acetylated by acetylsalicylic acid (aspirin) initiates the biosynthesis of a LXA4 receptor ligand. We therefore determined whether colonic cell lines (HT-29 Cl.19A, Caco-2, or T84) express the COX II isozyme. Results for RT-PCR and Western blot analysis showed that COX I as well as an IL-1beta- and TNF-alpha-inducible COX II are expressed in HT-29 Cl.19A. In addition, aspirin-treated enterocytes generated 15R-HETE, a precursor of 15-epi-LXA4 biosynthesis, whose potent bioactions were mimicked by the stable analog 15R/S-methyl-LXA4. Taken together, these results identify an endogenous pathway for downregulating mucosal inflammatory events and suggest a potential therapeutic benefit for LXA4 stable analogs.

Migration of neutrophils through epithelial monolayers.

Acute bacterial infections are a major challenge to epithelial linings that interface indirectly with the external world. Such infections are in part fought by neutrophils, which phagocytose and destroy pathogens. Neutrophils arrive at the infection sites by emigrating from small blood vessels and subsequently interacting with and transmigrating across columnar epithelia. There is substantial interest in defining the mechanisms and functional consequences of neutrophil-epithelial interactions. Model systems reveal that specific molecular events are required for appropriate neutrophil-epithelial interactions and, as a result of these interactions, neutrophils may reversibly modulate diverse epithelial functions.

Increases in guinea pig small intestinal transepithelial resistance induced by osmotic loads are accompanied by rapid alterations in absorptive-cell tight-junction structure.

In some epithelia, mucosal exposure to osmotic loads produces an increase in transepithelial resistance that is presumed to relate to the collapse of the paracellular spaces. Since proximal small intestinal epithelium may transiently encounter osmotic loads during normal digestion, we examined the short-term effect of osmotic loads on resistance and on epithelial structure of mucosal sheets prepared from guinea pig jejunum using Ussing-chamber, thin-section electron-microscopic, and freeze-fracture techniques. After equilibration of mucosal sheets in chambers, mucosal buffer tonicity was increased to 600 mosM with mannitol. This resulted in a 64% increase in resistance within 20 min. Concomitantly, 600 mosM produced a decrease in tight-junction cation selectivity as judged from dilution potentials, collapse of paracellular spaces, decreased cytoplasmic electron density in 10-40% of absorptive cells, and focal absorptive-cell subjunctional lateral-membrane evaginations often associated with microfilament arrays. Freeze-fracture replicas of absorptive-cell tight junctions revealed significant increases in both strand count and depth. Preincubation with 5 micrograms/ml cytochalasin D reduced the 600 mosM resistance increase caused by 600 mosM exposure by 48% but did not prevent the collapse of paracellular spaces. Lowered temperatures that produced morphologic evidence consistent with a gel-phase transition of absorptive-cell lateral membranes prevented both the resistance response and the alterations in tight-junction structure. In conclusion, transient osmotic loads produce an increase in resistance in jejunal epithelium and alter both absorptive-cell tight-junction charge selectivity and structure. These responses, which may have physiologic implications, can be reduced by cytoskeletal inhibitors and ablated by conditions that restrict mobility of absorptive-cell lateral-membrane molecules.

Occluding junction structure-function relationships in a cultured epithelial monolayer.

Electrical circuit analysis was used to study the structural development of occluding junctions (OJs) in cultured monolayers composed to T84 cells. The magnitude of the increments in transepithelial resistance predicted by such analysis was compared with the magnitude of the measured increments in resistance. Confluent sheets of epithelial cells were formed after cells were plated at high density on collagen-coated filters. Using Claude's OJ strand count-resistance hypothesis (1978, J. Membr. Biol. 39:219-232), electrical circuit analysis of histograms describing OJ strand count distribution at different time points after plating predicted that junctional resistance should rise in a proportion of 1:21:50 from 18 h to 2 d to 5 d. This reasonably paralleled the degree of rise in transepithelial resistance over this period, which was 1:29:59. The ability to predict the observed resistance rise was eliminated if only mean strand counts were analyzed or if electrical circuit analysis of OJ strand counts were performed using an OJ strand count-resistance relationship substantially different from that proposed by Claude. Measurements of unidirectional fluxes of inulin, mannitol, and sodium indicated that restriction of transjunctional permeability accounted for the observed resistance rise, and that T84 junctional strands have finite permeability to molecules with radii less than or equal to 3.6 A but are essentially impermeable to molecules with radii greater than or equal to 15 A. The results suggest that general correlates between OJ structure and OJ ability to resist passive ion flow do exist in T84 monolayers. The study also suggests that such correlates can be obtained only if OJ structural data are analyzed as an electrical circuit composed of parallel resistors.

Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity.

Intestinal absorptive cells may modulate both the structure and function of occluding junctions by a cytoskeleton dependent mechanism (Madara, J. L., 1983, J. Cell Biol., 97:125-136). To further examine the putative relationship between absorptive cell occluding junctions and the cytoskeleton, we assessed the effects of cytochalasin D (CD) on occluding junction function and structure in guinea pig ileum using ultrastructural and Ussing chamber techniques. Maximal decrements in transepithelial resistance and junctional charge selectivity were obtained with 10 micrograms/ml CD and the dose-response curves for these two functional parameters were highly similar. Analysis of simultaneous flux studies of sodium and the nonabsorbable extracellular tracer mannitol suggested that CD opened a transjunctional shunt and that this shunt could fully account for the increase in sodium permeability and thus the decrease in resistance. Structural studies including electron microscopy of detergent-extracted cytoskeletal preparations revealed that 10 micrograms/ml CD produced condensation of filamentous elements of the peri-junctional contractile ring and that this was associated with brush border contraction as assessed by scanning electron microscopy. Quantitative freeze-fracture studies revealed marked aberrations in absorptive cell occluding junction structure including diminished strand number, reduced strand-strand cross-linking, and failure of strands to impede the movement of intramembrane particles across them. In aggregate these studies show that CD-induced perturbation of the absorptive cell cytoskeleton results in production of a transepithelial shunt which is fully explained by a defect in the transjunctional pathway. Furthermore, substantial structural abnormalities in occluding junction structure accompany this response. Lastly, the abnormalities in occluding junction structure and function coincide with structural changes in and contraction of the peri-junctional actin-myosin ring. These data suggest that a functionally relevant association may exist between the cytoskeleton and the occluding junction of absorptive cells. We speculate that such an association may serve as a mechanism by which absorptive cells regulate paracellular transport.

Neutrophil migration across a cultured epithelial monolayer elicits a biphasic resistance response representing sequential effects on transcellular and paracellular pathways.

Migration of polymorphonuclear leukocytes across epithelia is a hallmark of many inflammatory disease states. Neutrophils traverse epithelia by migrating through the paracellular space and crossing intercellular tight junctions. We have previously shown (Nash, S., J. Stafford, and J.L. Madara. 1987. J. Clin. Invest. 80:1104-1113), that leukocyte migration across T84 monolayers, a model human intestinal epithelium, results in enhanced tight junction permeability--an effect quantitated by the use of a simple, standard electrical assay of transepithelial resistance. Here we show that detailed time course studies of the transmigration-elicited decline in resistance has two components, one of which is unrelated to junctional permeability. The initial decrease in resistance, maximal 5-13 min after initiation of transmigration, occurs despite inhibition of transmigration by an antibody to the common beta subunit of neutrophil beta 2 integrins, and is paralleled by an increase in transepithelial short-circuit current. Chloride ion substitution and inhibitor studies indicate that the early-phase resistance decline is not attributable to an increase in tight junction permeability but is due to decreased resistance across epithelial cells resulting from chloride secretion. Since T84 cells are accepted models for studies of the regulation of Cl- and water secretion, our results suggest that neutrophil transmigration across mucosal surfaces (for example, respiratory and intestinal tracts) may initially activate flushing of the surface by salt and water. Equally important, these studies, by providing a concrete example of sequential transcellular and paracellular effects on transepithelial resistance, highlight the fact that this widely used assay cannot simply be viewed as a direct functional probe of tight junction permeability.

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)

Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic.

The massive secretion of salt and water in cholera-induced diarrhea involves binding of cholera toxin (CT) to ganglioside GM1 in the apical membrane of intestinal epithelial cells, translocation of the enzymatically active A1-peptide across the membrane, and subsequent activation of adenylate cyclase located on the cytoplasmic surface of the basolateral membrane. Studies on nonpolarized cells show that CT is internalized by receptor-mediated endocytosis, and that the A1-subunit may remain membrane associated. To test the hypothesis that toxin action in polarized cells may involve intracellular movement of toxin-containing membranes, monolayers of the polarized intestinal epithelial cell line T84 were mounted in modified Ussing chambers and the response to CT was examined. Apical CT at 37 degrees C elicited a short circuit current (Isc: 48 +/- 2.1 microA/cm2; half-maximal effective dose, ED50 integral of 0.5 nM) after a lag of 33 +/- 2 min which bidirectional 22Na+ and 36Cl- flux studies showed to be due to electrogenic Cl- secretion. The time course of the CT-induced Isc response paralleled the time course of cAMP generation. The dose response to basolateral toxin at 37 degrees C was identical to that of apical CT but lag times (24 +/- 2 min) and initial rates were significantly less. At 20 degrees C, the Isc response to apical CT was more strongly inhibited (30-50%) than the response to basolateral CT, even though translocation occurred in both cases as evidenced by the formation of A1-peptide. A functional rhodamine-labeled CT-analogue applied apically or basolaterally at 20 degrees C was visualized only within endocytic vesicles close to apical or basolateral membranes, whereas movement into deeper apical structures was detected at 37 degrees C. At 15 degrees C, in contrast, reduction to the A1-peptide was completely inhibited and both apical and basolateral CT failed to stimulate Isc although Isc responses to 1 nM vasoactive intestinal peptide, 10 microM forskolin, and 3 mM 8Br-cAMP were intact. Re-warming above 32 degrees C restored CT-induced Isc. Preincubating monolayers for 30 min at 37 degrees C before cooling to 15 degrees C overcame the temperature block of basolateral CT but the response to apical toxin remained completely inhibited. These results identify a temperature-sensitive step essential to apical toxin action on polarized epithelial cells. We suggest that this event involves vesicular transport of toxin-containing membranes beyond the apical endosomal compartment.

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.

Surface attachment of Salmonella typhimurium to intestinal epithelia imprints the subepithelial matrix with gradients chemotactic for neutrophils.

During intestinal disease induced by Salmonella typhimurium transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane. Among the events stimulated by these interactions is the release of chemotaxins that guide PMN through the subepithelial matrix and subsequently through the epithelium itself (McCormick, B.A., S.P. Colgan, C. Delp-Archer, S.I. Miller, and J.L. Madara. 1993. J. Cell Biol. 123:895-907). Given the substantial volume flow that normally characterizes matrix compartments underlying transporting epithelia, it is unclear how such transmatrix signaling is sustained. Here we show that when underlying matrices are isolated from biophysically confluent polarized monolayers of the human intestinal epithelial cell line T84, they fail to support substantial transmatrix migration of PMN unless an exogenous chemotactic gradient is imposed. In contrast, such matrices isolated from confluent monolayers apically colonized with S. typhimurium support spontaneous transmatrix migration of PMN. Such chemotactic imprinting of underlying matrices is resistant to volume wash and is paralleled by secretion of the known matrix-binding chemokine IL-8. Chemotactic imprinting of the matrix underlying S. typhimurium-colonized monolayers is dependent on epithelial protein synthesis, is directional implying the existence of a gradient, and is neutralized by antibodies either to IL-8 or to the IL-8 receptor on PMN. An avirulent S. typhimurium strain, PhoPc, which attaches to epithelial cells as efficiently as wild-type S. typhimurium, fails to induce basolateral secretion of IL-8 and likewise fails to imprint matrices. Together, these observations show that the epithelial surface can respond to the presence of a luminal pathogen and subsequently imprint the subepithelial matrix with retained IL-8 gradients sufficient to resist washout effects of the volume flow that normally traverses this compartment. Such data further support the notion that the primary role for basolateral secretion of IL-8 by the intestinal and likely other epithelia is recruitment of PMN through the matrix to the subepithelial space, rather than directing the final movement of PMN across the epithelium.

CD47 mediates post-adhesive events required for neutrophil migration across polarized intestinal epithelia.

Transepithelial migration of neutrophils (PMN) is a defining characteristic of active inflammatory states of mucosal surfaces. The process of PMN transepithelial migration, while dependent on the neutrophil beta 2 integrin CD11b/CD18, remains poorly understood. In these studies, we define a monoclonal antibody, C5/D5, raised against epithelial membrane preparations, which markedly inhibits PMN migration across polarized monolayers of the human intestinal epithelial cell line T84 in a bidirectional fashion. In T84 cells, the antigen defined by C5/D5 is upregulated by epithelial exposure to IFN-gamma, and represents a membrane glycoprotein of approximately 60 kD that is expressed on the basolateral membrane. While transepithelial migration of PMN was markedly inhibited by either C5/D5 IgG or C5/D5 Fab fragments, the antibody failed to inhibit both adhesion of PMN to T84 monolayers and adhesion of isolated T84 cells to the purified PMN integrin, CD11b/CD18. Thus, epithelial-PMN interactions blocked by C5/D5 appear to be downstream from initial CD11b/CD18-mediated adhesion of PMN to epithelial cells. Purification, microsequence analysis, and cross-blotting experiments indicate that the C5/D5 antigen represents CD47, a previously cloned integral membrane glycoprotein with homology to the immunoglobulin superfamily. Expression of the CD47 epitope was confirmed on PMN and was also localized to the basolateral membrane of normal human colonic epithelial cells. While C5/D5 IgG inhibited PMN migration even in the absence of epithelial, preincubation of T84 monolayers with C5/D5 IgG followed by antibody washout also resulted in inhibition of transmigration. These results suggest the presence of both neutrophil and epithelial components to CD47-mediated transepithelial migration. Thus, CD47 represents a potential new therapeutic target for downregulating active inflammatory disease of mucosal surfaces.

Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL.

Vibrio cholerae and Escherichia coli heat labile toxins (CT and LT) elicit a secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1) and subsequently activating basolateral effectors (adenylate cyclase). We have recently proposed that signal transduction in polarized cells may require transcytosis of toxin-containing membranes (Lencer, W. I., G. Strohmeier, S. Moe, S. L. Carlson, C. T. Constable, and J. L. Madara. 1995. Proc. Natl. Acad. Sci. USA. 92:10094-10098). Targeting of CT into this pathway depends initially on binding of toxin B subunits to GM1 at the cell surface. The anatomical compartments in which subsequent steps of CT processing occur are less clearly defined. However, the enzymatically active A subunit of CT contains the ER retention signal KDEL (RDEL in LT). Thus if the KDEL motif were required for normal CT trafficking, movement of CT from the Golgi to ER would be implied. To test this idea, recombinant wild-type (wt) and mutant CT and LT were prepared. The COOH-terminal KDEL sequence in CT was replaced by seven unrelated amino acids: LEDERAS. In LT, a single point mutation replacing leucine with valine in RDEL was made. Wt and mutant toxins displayed similar enzymatic activities and binding affinities to GM1 immobilized on plastic. Biologic activity of recombinant toxins was assessed as a Cl- secretory response elicited from the polarized human epithelial cell line T84 using standard electrophysiologic techniques. Mutations in K(R)DEL of both CT and LT delayed the time course of toxin-induced Cl- secretion. At T1/2, dose dependencies for K(R)DEL-mutant toxins were increased > or = 10-fold. KDEL-mutants displayed differentially greater temperature sensitivity. In direct concordance with a slower rate of signal transduction. KDEL-mutants were trafficked to the basolateral membrane more slowly than wt CT (assessed by selective cell surface biotinylation as transcytosis of B subunit). Mutation in K(R)DEL had no effect on the rate of toxin endocytosis. These data provide evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that both toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.

Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia.

In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and perhaps ER (Lencer, W.I., C. Constable, S. Moe, M. Jobling, H.M. Webb, S. Ruston, J.L. Madara, T. Hirst, and R. Holmes. 1995. J. Cell Biol. 131:951-962). In this study, we tested whether CT's apical membrane receptor ganglioside GM1 acts specifically in toxin action. To do so, we used CT and the related Escherichia coli heat-labile type II enterotoxin LTIIb. CT and LTIIb distinguish between gangliosides GM1 and GD1a at the cell surface by virtue of their dissimilar receptor-binding B subunits. The enzymatically active A subunits, however, are homologous. While both toxins bound specifically to human intestinal T84 cells (Kd approximately 5 nM), only CT elicited a cAMP-dependent Cl- secretory response. LTIIb, however, was more potent than CT in eliciting a cAMP-dependent response from mouse Y1 adrenal cells (toxic dose 10 vs. 300 pg/well). In T84 cells, CT fractionated with caveolae-like detergent-insoluble membranes, but LTIIb did not. To investigate further the relationship between the specificity of ganglioside binding and partitioning into detergent-insoluble membranes and signal transduction, CT and LTIIb chimeric toxins were prepared. Analysis of these chimeric toxins confirmed that toxin-induced signal transduction depended critically on the specificity of ganglioside structure. The mechanism(s) by which ganglioside GM1 functions in signal transduction likely depends on coupling CT with caveolae or caveolae-related membrane domains.

Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination.

Epithelia of the vertebrate intestinal tract characteristically maintain an inflammatory hyporesponsiveness toward the lumenal prokaryotic microflora. We report the identification of enteric organisms (nonvirulent Salmonella strains) whose direct interaction with model human epithelia attenuate synthesis of inflammatory effector molecules elicited by diverse proinflammatory stimuli. This immunosuppressive effect involves inhibition of the inhibitor kappaB/nuclear factor kappaB (IkappaB/NF-kappaB) pathway by blockade of IkappaB-alpha degradation, which prevents subsequent nuclear translocation of active NF-kappaB dimer. Although phosphorylation of IkappaB-alpha occurs, subsequent polyubiquitination necessary for regulated IkappaB-alpha degradation is completely abrogated. These data suggest that prokaryotic determinants could be responsible for the unique tolerance of the gastrointestinal mucosa to proinflammatory stimuli.

Interferon-gamma directly affects barrier function of cultured intestinal epithelial monolayers.

Although epithelia, which often are in intimate contact with lymphoid cells, may bear receptors for various cytokines, it is unclear whether cytokines directly effect epithelial function. We examine the effects of the cytokine interferon (IFN) on barrier function of cultured monolayers of the T84 human intestinal epithelial cell line. Gamma IFN, in concentrations and exposures required to show its other biological effects, directly affects such monolayers. Monolayer resistance is substantially diminished by gamma IFN. Such effects were not due to cytotoxicity as judged morphologically and by LDH assays. Solute fluxes and dual Na+-mannitol flux analysis indicate that the resistance decrease is due to an effect of gamma IFN on tight junction permeability. The effects of gamma IFN on monolayer barrier function were not duplicated by the cytokines interleukin 1, interleukin 2, or tumor necrosis factor. We speculate that such products of activation of lymphoid cells might influence barrier function of intestinal, and perhaps other epithelia in disease states.

Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells.

Closure of superficial wounds in epithelia occurs by migration of cells shouldering the wound. We describe an in vitro model of such restitution using a human intestinal epithelial cell line, T84. T84 cells were grown on novel optically transparent type 1 collagen membranes without underlying filter supports. Monolayers so grown display substantial barrier function (400-500 ohm.cm2; 1.3 +/- 0.4 nmol.h-1.cm-2 mannitol flux). Wounds made with micropipettes were accompanied by a fall in resistance and rise in monolayer permeability to mannitol and inulin. After injury, cells shouldering wounds migrated, by extension of lamellipodia-like processes, to reseal wounds as defined by structural and functional criteria. F actin arcs crossed the base of the lamellipodia-like extensions and F actin microspikes projected from the leading edge of these extensions. Villin, an epithelial-specific cytoskeletal protein with both F actin bundling and severing capacities, was also expressed at the leading edge in a pattern consistent with a regulatory role in the dynamic restructuring of lamellipodia. Lastly, myosin II was predominantly localized to the basal regions of lamellipodia, though occasional staining was seen close to the advancing edge. Myosin I, a recently recognized myosin family member considered to be essential for fibroblast and slime mold motility, was present throughout lamellipodia in punctate fashion, but was not concentrated at the leading edge.

Structural and functional evolution of jejunal allograft rejection in rats and the ameliorating effects of cyclosporine therapy.

We assessed the structural and functional evolution of small intestinal transplant rejection in a rat model by use of 1-micron section, electron microscopic, and in vitro electrophysiologic techniques to study jejunal mucosa 3, 6, and 9 d posttransplantation. The earliest structural abnormalities detected in jejunal loops transplanted from Lewis X Brown Norway F1 hybrids into Lewis rats occurred within 3 d posttransplantation and consisted of focal endothelial cell injury of the microvasculature and focal injury of crypt epithelial cells. Both alterations were associated with adjacent infiltration of large lymphoid cells, and both markedly progressed and became rather diffuse over the following 6 d. In contrast, villus absorptive cells were not markedly altered in structure until the 9th postoperative day. As compared with host jejuna, allograft jejunal epithelium demonstrated multiple functional abnormalities. Transepithelial resistance declined progressively by days 6 and 9 (both P less than 0.05), although baseline transepithelial spontaneous potential difference was only affected at day 9 (P less than 0.01). Stimulated absorption by allograft jejuna, as assessed by measuring electrical response to mucosal glucose, was not significantly diminished until day 9 (P less than 0.05). In contrast, stimulated secretion assessed by measurement of electrical response to serosal theophylline was diminished by day 6 (P less than .01). These data suggest that the earliest epithelial injury during rejection, as judged both structurally and functionally, occurs in the crypt and is paralleled by endothelial injury at the level of the microvasculature. Thus, the primary targets for rejection are most likely endothelial cells and crypt epithelial cells. In contrast, structural and functional impairment of villus epithelium is detectable only at substantially later times during rejection and are most likely secondary processes related to either ischemia produced by microvascular injury or decreased epithelial regenerative ability secondary to crypt injury. Last, we show that the detrimental structural and functional sequellae of jejunal transplantation across the major histocompatibility complex in this model is strikingly ameliorated with cyclosporine therapy.

Effects of polymorphonuclear leukocyte transmigration on the barrier function of cultured intestinal epithelial monolayers.

We describe a model to study the effects of polymorphonuclear leukocyte (PMN) transmigration on the intestinal epithelial barrier. Human PMN were induced to transmigrate across high resistance monolayers of a cultured human intestinal epithelial cell line (T84 cells) by chemotactic gradients produced by formyl methionyl leucyl phenylalanine (FMLP). With maximal transmigration monolayer resistance decreased by 48 +/- 12.6% in 15 min and by 83 +/- 1.6% in 60 min. This response was dependent on the size of the FMLP gradient and the density of PMN transmigration. The decrease in resistance correlated with number of PMN migrating across monolayers, and was accompanied by increases in flux of paracellular tracers. Macromolecular tracer studies localized the leak sites to foci at which PMN impaled the epithelium. Removal of the chemotactic gradient led to restoration of baseline resistance within 18 h. PMN transmigration across intestinal epithelial monolayers occurs via intercellular occluding junctions and may be associated with a reversible increase in epithelial permeability.

Supraphysiologic L-tryptophan elicits cytoskeletal and macromolecular permeability alterations in hamster small intestinal epithelium in vitro.

We have previously shown that Na(+)-coupled transport of glucose and amino acids across the apical membrane of intestinal absorptive cells is accompanied by alterations in cytoskeletal structure and altered sieving of small hydrophilic solutes by tight junctions. Here we report that in response to the essential amino acid L-tryptophan at lumenal concentrations likely to be supraphysiological (1 mM or greater), these responses are so exaggerated as to induce disruption of tight junctions and transepithelial macromolecular leaks. Since these effects of L-tryptophan are energy and Na+ dependent and occur with mucosal but not serosal exposure to L-tryptophan, it appears they are triggered by activation of a Na(+)-nutrient cotransporter in the apical membrane of absorptive cells rather than by the presence of an unidentified trace contaminant. Our findings suggest the possibility that dietary supplementation by L-tryptophan may result in loss of the intestinal epithelial barrier to dietary antigens. We speculate that such a response to supraphysiologic tryptophan may contribute, in part, to the recently recognized curious tryptophan-induced eosinophilia myalgia syndrome.

Microfilament-dependent activation of Na+/K+/2Cl- cotransport by cAMP in intestinal epithelial monolayers.

cAMP-mediated stimulation of Cl- secretion in the human intestinal cell line T84 is accompanied by significant remodeling of F-actin, and both the secretory and cytoskeletal responses may be largely ablated by previous cell loading with phalloidin derivatives, reagents that prevent dynamic reordering of microfilaments (1991. J. Clin. Invest. 87:1903-1909). In this study, we examined the effect of phalloidin loading on the cAMP-elicited activity of the individual membrane-associated transport proteins involved in electrogenic Cl- secretion. Efflux of 125I and 86Rb was used to assay forskolin-stimulated Cl- and K+ conductances, respectively, and no inhibitory effect of phalloidin could be detected. Na+/K(+)-ATPase pump activity, assessed as bumetanide-insensitive 86Rb uptake and the ability of monolayers to generate a Na+ absorptive current in response to apical addition of a Na+ ionophore, was not different between control and phalloidin-loaded monolayers. Forskolin was found to stimulate Na+/K+/2Cl- cotransport (bumetanide-sensitive 86Rb uptake) in time-dependent fashion. In the absence of any agonist, cotransporter activity was markedly decreased in phalloidin-loaded monolayers. Furthermore, under phalloidin-loaded conditions, the forskolin-elicited increase in bumetanide-sensitive 86Rb uptake was markedly attenuated. These findings suggest that cAMP-induced activity of Cl- channels, K+ channels, and the Na+/K(+)-ATPase are not influenced by F-actin stabilization. However, cAMP-induced activation of the Na+/K+/2Cl- cotransporter appears to be microfilament-dependent, and ablation of this event is likely to account for the inhibition of cAMP-elicited Cl- secretion seen in the phalloidin-loaded state. Such findings suggest that Na+/K+/2Cl- cotransporter is functionally linked to the cytoskeleton and is a regulated site of cAMP-elicited electrogenic Cl- secretion.