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 ofcolumnar epithelia, is regulated by rho. and basolateral membranes, high transepithelial resistance to passive ion flow, and a Cl-secretory pathway analogous to that found in native intestinal crypt epithelium. We have also determined the effects of rhoC overexpression in intestinal epithelial cell lines. Our data indicate that rho plays an important regulatory role in determining microfilament organization in the apical pole of columnar epithelial cells and, in so doing, influences the associated tight junction and epithelial permeability. Concurrent with these effects, ZO-1, a tight junction structural element, moves off the membrane, while E-cadherin, the sealing element of the closely related adherens junction, is preserved on the membrane.The rho proteins are members of a large family of small GTP-binding proteins (21 kDa) that are believed to be involved in regulating assembly of the actin cytoskeleton. In unpolarized mammalian cells, rho proteins (rhoA, B, and C) regulate assembly of focal adhesions and basal filamentous (F) actin stress fibers (1). The Clostridium botulinum toxin C3 transferase selectively blocks rho-effector coupling by ADPribosylation of rho on Asn-41 (2-4) and has been a useful tool in examining the biological function of rho proteins. In fibroblasts, C3 transferase induces disassembly of stress fibers that are localized at the base of cells (3, 5). Conversely, microinjection of a constitutively activated form of rho into quiescent serum-starved cells results in the appearance of prominent stress fibers and focal adhesions at the cell-matrix interface (1, 6).Polarized columnar epithelial cells, such as those lining the alimentary tract, airways, and renal tubules, differ markedly from spreading unpolarized cells in which rho biology has largely been studied. For example, intestinal epithelial cells maintain a tall cylindrical form, have apical intercellular tight junctions that serve as barriers to restrict paracellular permeability, and exhibit morphologically defined subdomains of cytoskeletal structure. Stable lateral and basal F-actin filaments form a submembrane cortex thought to assist in maintaining the cylindrical shape of columnar epithelial cells and in anchoring a variety of basolateral membrane proteins. In contrast, the apical pole of polarized epithelial cells consists of a perijunctional tensile ri...

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.

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)

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.