Clin. Invest. 1991. 88:1605-1612

Neutrophil transmigration across intestinal epithelia is thought to contribute to epithelial dysfunction and characterizes many inflammatory intestinal diseases. Neutrophils activated by factors, normally present in the lumen, release a neutrophilderived secretagogue activity to which intestinal epithelia respond with an electrogenic chloride secretion, the transport event which underlies secretory diarrhea. Using sequential ultrafiltration, column chromatographic, and mass and Raman spectroscopic techniques, neutrophil-derived secretagogue was identified as 5'-AMP. Additional studies suggested that neutrophil-derived 5'-AMP is subsequently converted to adenosine at the epithelial cell surface by ecto-5'-nucleotidase and that adenosine subsequently activates intestinal secretion through adenosine receptors on the apical membrane of target intestinal epithelial cells. These findings suggest that this ATP metabolite may serve as a neutrophil-derived paracrine mediator that contributes to secretory diarrhea in states of intestinal inflammation. (J. Clin. Invest. 1993.91:2320-2325

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,

Abstract. 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 A~-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 A~-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 1'84 were mounted in modified Ussing chambers and the response to CT was examined. Apical CT at 37~ elicited a short circuit current (Isc: 48 • 2.1/zA/cm2; half-maximal effective dose, ED50 •0.5 nM) after a lag of 33 • 2 rain which bidirectional ~Na + and ~C1-flux studies showed to be due to electrogenic C1-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~ was identical to that of apical CT but lag times (24 • 2 rain) and initial rates were significantly less. At 20~ 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 A~-peptide. A functional rhodamine-labeled CT-analogue applied apically or hasolateraUy at 20~ was visualized only within endocytic vesicles close to apical or basolateral membranes, whereas movement into deeper apical structures was detected at 37~ At 15~ in contrast, reduction to the Arpeptide was completely inhibited and both apical and basolateral CT failed to stimulate Isc although Isc responses to 1 nM vasoactive intestinal peptide, 10 #M forskolin, and 3 raM 8Br-cAMP were intact. Re-warming above 32~ restored CT-induced Isc. Preincubating monolayers for 30 rain at 37~ before cooling to 15~ 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.

Abstract. 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. . Z 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-,), on PMN migration across cultured monolayers of the human intestinal epithelial cell line Tu. Transepithelial migration of PMN was initially assessed in the apicalto-basolateral direction, since previous studies indicate general qualitative similarities between PMN migration in the apical-to-basolateral and in the basolateralto-apical directions. In the apical-to-basolateral direction, epithelial exposure to IFN-.,/rnarkedly upregulated transepithelial migration of PMN in a dose-and timedependent fashion as measured by both electrical and myeloperoxidase assays. This IFN-~/-elicited effect on transmigration was specifically due to a IFN-,/effect on epithelial cells and was not secondary to IFN-,y effects on epithelial tight junction permeability. Moreover, this IFN-.y effect was dependent on epithelial protein synthesis, and involved a pathway in which CDllb/18, but not ICAM-1 or CDlla/18, appeared to play a crucial role in PMN-epithelial adhesion. IFN-~/also substantially modified PMN transepithelial migration in the natural, basolateral-to-apical direction. The IFN-'y effect on naturally directed transmigration was also specifically due to an IFN-3, effect on epithelial cells, showed comparable time and dose dependency to that of oppositely directed migration, was CDllb/18 dependent, and required epithelial protein synthesis. Additionally, however, important qualitative differences existed in how IFN-3, affected transmigration in the two directions. In contrast to apical-to-basolateral directed migration, IFN-,), 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-3, 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. Specifically, it appears that in naturally directed migration, IFN-~/ may enhance the retention time of the recruited PMN in the paracellular space below tight junctions, and does so, at least in part, by downregulating a PMN-epithelial interactive event req...

Abstract. 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 TM 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 transmigrationelicited 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/32 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 1"84 cells are accepted models for studies of the regulation of C1-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.

SUMMARY:Typically, electrophysiotogic studies of epithelial monolayers are either performed in formahzed Ussing chamber systems which yield highly accurate results or in simple setups using recording devices which have limitations in accuracy or the range of measurements which can be performed or both. Here we detail a simple method of interfacing traditionally accurate Ussing chambers systems with commercially available filter supports on which epithelial monolayers can be grown. We also detail simple methods for growing inverted monolayers, suitable for electrophysiologic assays, for use in studies where cells or particles must be layered by gravity on the undersurface (basolateral pole) of the filter. Both approaches allow experiments to be performed on large numbers of monolayers synchronously. As an example of the use of this system, we analyze the sequelae of neutrophil migration across monolayers of the intestinal cell line T84. Neutrophil migration across monolayers can occur in either direction, is dependent on neutrophil surface ~2 integrins, and is paralleled by a decrease in epithelial barrier function as detected electrically. We have previously shown in formal Ussing chamber studies that neutrophil-epithelial interactions elicit a modest short-circuit current indicative of electrogenic ion transport. We show here that this short-circuit current response can be readily detected using the simple approach described.

No abstract