THEME
I. Neutrophil adhesive interactions with intestinal epithelium*

Charles A. Parkos

Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322

    ABSTRACT
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In many inflammatory conditions of the gastrointestinal tract, disease activity and patient symptoms correlate with the histological finding of neutrophil (PMN) migration across the epithelium. PMN interactions with intestinal epithelium can influence epithelial functions ranging from barrier maintenance to electrolyte secretion. Additionally, PMN recruitment to the epithelium can be modulated by epithelial interactions with luminal enteric pathogens. Adhesive interactions between PMN and intestinal epithelial cells have been shown to be distinct from interactions of PMN with endothelia. In particular, PMN transepithelial migration is modulated by a distinct array of cytokines including interferon-gamma and interleukin-4 and requires the PMN beta 2-integrin CD11b/CD18 but is independent of CD11a/CD18, selectins, and intercellular adhesion molecule 1. Additionally, an integral membrane protein termed CD47 has recently been shown to play an important role in PMN transepithelial migration at point(s) subsequent to initial adhesive interactions. This article provides a brief overview of PMN interactions with epithelia and their functional consequences in relation to inflammatory disease.

interferon-gamma ; CD47; CD11b/CD18; selectins; inflammation; intercellular adhesion molecule 1; transepithelial migration

    ARTICLE
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UNDER MOST CIRCUMSTANCES, active inflammatory diseases of the gastrointestinal tract are characterized by neutrophil (PMN) infiltrates within the mucosa and surface epithelium. PMN migration across gut epithelium is a key event because it results in organ dysfunction and disease symptoms. Disorders associated with PMN transepithelial migration include ulcerative colitis, Crohn's disease, bacterial enterocolitis, Helicobacter gastritis, cholangitis, acute cholecystitis, and many others. In cases of inflammatory bowel disease, characterized by a chronic waxing and waning course of debilitating symptoms, there are neutrophilic infiltrates within the colonic epithelium of biopsies taken during active disease periods (Fig. 1A). PMN are also a central component in intestinal dysfunction after ischemic injury and are largely responsible for subsequent increased mucosal permeability and fluid loss into the lumen. Thus, although PMN comprise the immune system's first line of defense, PMN migration in the gut has functional consequences that correlate with disease symptoms.


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Fig. 1.   Neutrophil (PMN) migration across intestinal epithelium. A: section of colonic mucosal biopsy from a patient with active ulcerative colitis. PMN are observed migrating across the epithelium (E) and collecting within the crypt lumen (L). B: section of a model intestinal epithelial monolayer (T84 cells) obtained during a PMN transmigration assay. Neutrophils are observed migrating across the epithelium and porous filter support in response to a transepithelial gradient of the chemotactic peptide N-formyl-Met-Leu-Phe. Magnification, ×400.

A number of studies have shed light on understanding the functional consequences of PMN interactions with intestinal epithelium. A model for assessing PMN interactions with epithelial cells is shown in Fig. 1B. Here, PMN are observed migrating across a monolayer of T84 intestinal epithelial cells in response to a transepithelial gradient of the chemotactic peptide N-formyl-Met-Leu-Phe. An intuitively obvious consequence of PMN migration across an epithelium is the disruption of important barrier function. As might be expected, the magnitude of barrier dysfunction is related to the number of PMN migrating across the epithelium. However, this process is not a linear function. Whereas the passage of a single PMN across epithelial tight junctions results in rapid resealing with little loss of barrier function (22), large-scale PMN migration results in the formation of sizable epithelial discontinuities, which may be the precursors of epithelial erosions and ulcers characteristic of many inflammatory conditions. Such large-scale disruption of barrier function provides luminal agents access to the systemic circulation.

There are also effects of PMN on epithelial function that are mediated by release of factors and subsequent signaling events. PMN that have migrated into the colonic lumen are in direct contact with potent activating agents such as N-formylated peptides, which serve to stimulate the release of 5'-adenosine monophosphate (5'-AMP) (18). In the intestinal crypt, 5'-AMP is converted to adenosine, which activates electrogenic chloride secretion and passive water movement into the lumen---the basis of secretory diarrhea. Recently, it has been demonstrated that intestinal epithelial cells express on their apical surface the ecto-5'-nucleotidase CD73 and adenosine receptors of the A2b type (30, 31). Furthermore, epithelial CD73 is glycosylphosphatidylinositol linked and appears to localize in membrane microdomains termed detergent-insoluble glycolipid rafts (30), which are putatively involved in clustering of proteins for efficient transmembrane signaling. These recent findings serve to shed light on how signals derived from migrating PMN might be efficiently amplified to produce functional responses in the epithelium.

Under natural circumstances, PMN movement across an epithelium occurs along the basolateral epithelial membrane through the paracellular space before disruption of tight junctions. Because the paracellular space is of considerable length, often greater than 20 µm, multiple adhesive events must take place. Interestingly, it has been shown that PMN transmigration is 5-20 times more efficient in the natural, basolateral-to-apical direction compared with PMN migration in the apical-to-basolateral direction (26). Perhaps such findings are related to cortical restructuring of epithelial F actin (11), since these differences in efficiency of transmigration can be ablated by disruption of epithelial microfilaments. Tight junction function is intimately tied to the apical actin ring (17, 23), and events that alter microfilaments might facilitate PMN in gaining access to the tight junction, which serves as a rate-limiting factor in transmigration. Such results suggest complex signaling events between PMN and epithelial cells. Further evidence comes from recent studies involving PMN transendothelial migration (6). It was observed that disorganization of the endothelial cell-cell junctional components vascular E-cadherin, beta -catenin, and plakoglobin occurred in the vicinity of regions of firm adhesion between PMN and endothelial cells, and such findings correlated with increased permeability. Although such events have not been observed between PMN and epithelial cells, it is worth pointing out that transepithelial resistance, a reliable indicator of epithelial barrier function, is reversibly diminished during PMN transepithelial migration assays in the natural basolateral-to-apical direction in a fashion that precedes actual PMN migration and does not correlate with stimulated epithelial ion secretion (Ref. 26 and unpublished observations).

Molecules important in adhesive events between PMN and intestinal epithelial cells are summarized in Table 1. Adhesive molecules important in PMN interactions with vascular endothelium are included in Table 1 for comparison due to the wealth of information known about these interactions. As noted in Table 1, there are key differences between the adhesive events involved in PMN adhesive interactions between epithelial cells and endothelial cells. However, given the marked differences in the microenvironments of these two cellular barriers, it is logical to expect that there would be substantial differences in the adhesive interactions governing transendothelial and transepithelial migration.

                              
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Table 1.   Comparison of molecular requirements during PMN transendothelial and transepithelial migration

As shown in Table 1, beta 2-integrins are central in both PMN transendothelial and transepithelial migration. The importance of beta 2-integrins in PMN transepithelial migration is exemplified in experiments with PMN from patients with leukocyte adhesion deficiency (LAD), who specifically lack beta 2-integrins. Here, LAD PMN failed to migrate across monolayers of intestinal epithelial cells in the presence of a chemotactic gradient (26). The beta 2-integrins are heterodimeric integral membrane glycoproteins expressed only on leukocytes and consist of a common CD18 beta -chain that can associate with one of four alpha -chains termed CD11a (LFA-1), CD11b (Mac-1), CD11c (p150), and CD11d (see Ref. 29 for review). In experiments with blocking antibodies specific for beta 2-integrin alpha -chains, it was determined that, in contrast to interactions with endothelial cells, PMN adhesion to epithelial cells is mediated exclusively by CD11b/CD18 (26). Although such studies demonstrating the importance of CD11b/CD18 in PMN transepithelial migration have largely employed transformed epithelial cell lines, the findings have also recently been verified in adhesion experiments using isolates of normal human colonic epithelial cells and purified CD11b/CD18 (24). These findings were the first indication of the differences existing between PMN adhesive interactions with endothelia and epithelia and also served to justify the use of transformed intestinal epithelia in models of PMN-epithelial interactions.

As indicated in Table 1, selectins are important in PMN interactions with vascular endothelium. However, whereas selectins (CD62 E, P, L) mediate the initial adhesive interactions between PMN and endothelium and account for the phenomenon of PMN rolling (see Ref. 29 for review), transepithelial migration is not inhibited by antibodies against any of the selectins (4). Such results are not so surprising in light of the exquisite dependence of selectin-mediated events on fluid shear and the lack of it in transepithelial migration assays. However, there is evidence suggesting a role of carbohydrate-mediated interactions in PMN transepithelial migration. Certain simple or complex sugars such as mannose 6-phosphate, glucose 6-phosphate, fucoidin, and the yeast-derived phosphomannan oligosaccharide PPME are potent inhibitors of PMN transepithelial migration (4). Thus, although classic selectin-mediated adhesive interactions do not appear to be important in PMN-epithelial adhesive interactions, there may be a role for other, as yet undefined carbohydrate-based interactions.

Other important endothelial ligands for PMN do not appear to have a role in PMN-epithelial interactions. PECAM (CD31) (21), which colocalizes to endothelial cell-cell lateral junctions, has been shown to mediate transendothelial migration of both PMN and monocytes through homotypic and heterotypic adhesive interactions. However, PECAM does not appear to be expressed by colonic epithelial cells, and blocking CD31 antibodies have no effect on PMN transepithelial migration (unpublished observations). Another important PMN adhesive receptor on endothelial cells is intercellular adhesion molecule 1 (ICAM-1; CD54) (9), which is differentially expressed under inflammatory conditions (see Ref. 27 for review). ICAM-1 localizes to the luminal membrane of vascular endothelium and is thus poised to serve as an important counterreceptor for both CD11a/CD18 and CD11b/CD18 on adherent PMN. As outlined below, ICAM-1 expression on intestinal epithelium has unique features that raise questions regarding its role as a receptor for migrating PMN.

ICAM-1 merits further discussion as a potential epithelial counterreceptor for PMN (CD11b/CD18). Although it is now clear that ICAM-1 plays a critical role as a receptor for beta 2-integrins during PMN migration out of the vasculature, its subcellular distribution within epithelia precludes a role in PMN migration across the epithelium under natural (basolateral to apical) conditions. Specifically, intestinal epithelial cells do not normally express ICAM-1 in a basal state, but expression is induced to the apical membrane under inflammatory conditions (24) and after invasion by bacterial pathogens (12). Under such conditions, PMN can adhere to apically expressed ICAM-1 in a CD11b/CD18-dependent manner. However, the physiological relevance of this event is unclear, since transmigrating PMN would not have access to apically expressed ICAM-1 until after crossing the epithelium (24). The potential role of luminally expressed ICAM-1 is open for speculation. One possible role might be in holding inflammatory cells in or near sites of inflammation. Given luminal fluid flow, partly in response to electrogenic chloride secretion from PMN-derived 5'-AMP, ICAM-1 might serve as an adhesive tether to retain PMN at specific locations. This adhesive foothold might also aid in PMN-mediated clearance of pathogens from mucosal surfaces.

As in interactions with vascular endothelium, PMN adhesive interactions with epithelial cells are modulated by inflammatory mediators and tissue hypoxia-ischemia. Generally, such conditions are characterized by enhanced PMN adhesion, activation, and tissue destruction. Local factors such as nitric oxide (see Ref. 1 for review) and mast cell products (13) serve to modulate PMN responses within the microcirculation and interstitium. In addition, a number of cytokines can act to uniquely alter epithelial adhesive interactions with PMN. Potent modulators of PMN-epithelial interactions include interferon-gamma (IFN-gamma ) (3) and interleukin-4 (IL-4), whereas IL-1, lipopolysaccharide, tumor necrosis factor-alpha , and others strongly modulate PMN endothelial interactions (see Ref. 27 for review). In both cases, modulation is at least in part due to expression of new surface proteins. Epithelial proteins expressed after IFN-gamma or IL-4 stimulation are responsible for both enhanced and diminished PMN transepithelial migration, depending on the polarity of PMN migration (3). Upregulation of epithelial adhesive ligands also occurs after a period of tissue hypoxia (2). Transient exposure of epithelial monolayers to low oxygen tension results in significantly enhanced PMN transepithelial migration, which is CD11b/CD18 and protein synthesis dependent. Although the nature of these upregulated epithelial receptors remains obscure, they appear to be distinct from receptors upregulated on endothelial cells.

Other factors serve to modulate PMN interactions with surfaces and may contribute to the unique way(s) that PMN interact with epithelial cells. After extravasation, the environment of a migrating PMN changes from that of a moving fluid to one composed of a structured, three-dimensional protein matrix. In addition, during the process of migrating across the endothelium (toward an epithelium), alterations occur in the surface adhesive proteins on the PMN (see Ref. 29 for review) that likely serve to modulate subsequent adhesive interactions. For example, PMN responsiveness to agonist stimulation is altered by adherence to extracellular matrix components. Likewise, in the extracellular matrix, there is evidence for PMN adhesive interactions involving integrins other than of the beta 2 type. In particular, PMN lacking beta 2-integrins are able to migrate through an artificial matrix composed of collagen gels (28), and normal PMN have been shown to express adhesive integrins of the beta 1 and beta 3 types. However, the magnitude of the contributions of these other integrins to PMN-epithelial interactions may be minor, since functionally blocking antibodies against beta 1- and beta 3-integrins have no effect on PMN transepithelial migration (unpublished observations), and the majority of data indicate that integrins of the beta 2 type are the most important mediators of firm adhesive events between PMN and epithelial cells.

Studies over the past four years have shown that epithelial-derived signals can also direct PMN transepithelial migration. After invasion by bacterial pathogens, exposure to hypoxia, or stimulation with cytokines, the intestinal epithelium secretes potent chemoattractants such as IL-8 (2, 10, 19) from the basolateral surface (20) and other, as of yet incompletely characterized, agents from the apical surface (19, 20). Current evidence indicates that IL-8 is released from the basolateral aspect of the intestinal epithelium and may serve to "imprint" the underlying subepithelial matrix to establish a "fixed" gradient that decreases away from the epithelium (2, 20), thus serving to recruit PMN close to the basolateral aspect of the epithelium. To account for the enhanced transepithelial chemotactic response observed with epithelial invasion of pathogens such as Salmonella, transcellular chemotactic agent(s) released from the apical epithelial surface ultimately serve to drive PMN across the epithelium (19, 20).

Efforts aimed at characterizing proteins involved in PMN adhesive interactions with intestinal epithelium recently identified another membrane protein termed CD47 (25) (Table 1). Monoclonal antibodies were identified that were capable of completely blocking PMN migration across monolayers of T84 intestinal epithelial cells (25), vascular endothelium, and collagen-coated filters (5, 25). Characterization revealed the antigen to be CD47, a recently identified member of the immunoglobulin gene superfamily (16). The gene for CD47 encodes a protein of 305 amino acids. Several N-glycosylation sites, five transmembrane alpha -helixes, and an extracellular loop with homology to immunoglobulin V (IgV) are predicted from the primary structure.

Because CD47 is expressed on both epithelial cells and PMN, experiments have been performed to examine the relative contributions of epithelial vs. PMN CD47 in the transmigration response. These experiments revealed a central role for PMN CD47 in transmigration but also indicated a likely role for epithelial-derived CD47 (25). In vivo verification of the importance of CD47 in PMN migration was recently demonstrated with a mouse knockout model (15). Animals deficient in CD47 expression are viable but rapidly succumb after intra-abdominal challenge with Escherichia coli, which is at least partially due to delayed recruitment of PMN to the site of infection.

The mechanism of how CD47 influences PMN migration is not known, but the in vitro studies indicate that contributions of CD47 to PMN migration may be as important as those of the beta 2-integrins. Other studies have shown quite convincingly that CD47 can act to regulate alpha vbeta 3-integrin avidity for its ligand vitronectin (16). Molecular and biochemical analyses have revealed that CD47 directly associates with alpha vbeta 3 and that the extracellular IgV-like loop is critical for function. Although such studies provide an elegant mechanism of CD47 function, they do not explain the mechanism of CD47 regulation of PMN transepithelial migration. Specifically, as pointed out above, despite the fact that PMN express beta 3-integrins, functionally inhibitory anti-beta 3 monoclonal antibodies alone and in combination have no effect on PMN transepithelial migration (unpublished observations).

It is possible that CD47 may function directly as an adhesion receptor or serve a regulatory role in CD11b/CD18 function. As with CD31, perhaps CD47 is involved in homotypic or heterotypic adhesive interactions between PMN and epithelia. The structure of the extracellular loop would be consistent with that of an adhesion protein. However, this does not appear to be the case, since epithelial cells do not adhere to CD47 purified by different immunoaffinity techniques (unpublished observations). CD47 has also been reported to bind to thrombospondin, an adhesive glycoprotein that can mediate cell-cell and cell-matrix interactions. However, intestinal epithelium does not stain for thrombospondin, and antithrombospondin antibodies have no effect on PMN transepithelial migration (unpublished observations).

CD47 may also act by regulation of de-adhesion during CD11b/CD18-mediated transepithelial migration. Examples of regulated de-adhesion of integrins are not new and exist in other systems. It is possible that CD47 interacts directly with CD11b/CD18 within the plane of the membrane to regulate function. Another PMN membrane protein, the urokinase receptor (CD87), has been shown to modulate CD11b/CD18 function, apparently through direct interactions within the plane of the membrane. Indeed, recent studies show that inhibition of CD47-mediated transmigration results in accumulation of PMN within the epithelium (25), which would be consistent with this hypothesis.

From the evidence presented thus far, it is clear that PMN migration across epithelial surfaces has many unique features and indicates the likelihood of novel adhesive ligands awaiting discovery. Clues to the nature of epithelial counterreceptors might be had by utilizing extensive information regarding known ligands for CD11b/CD18. For example, recent studies demonstrate that the I domain, a stretch of 200 amino acids toward the amino-terminal end of the extracellular portion of CD11b, directly mediates binding of a number of ligands, including iC3b, fibrinogen, ICAM-1, ICAM-2, heparin, and a hookworm-derived PMN inhibitory factor (see Refs. 8, 14, and 32 for review). CD11b also binds to the blood coagulation factor X, but does so outside of the I domain. Despite the long list of known ligands for CD11b/CD18, none appear to mediate PMN transepithelial migration.

On the basis of these observations, what structures might be candidate epithelial ligands? As outlined above, evidence exists for a role of carbohydrate structures in PMN-epithelial adhesive interactions. Because heparin and heparan sulfate can bind to CD11b/CD18 (7), candidate ligands might include members of the proteoglycan family, which contain heparin or heparan sulfate moieties such as syndecan, glycipan, and perlecan. It is also likely that more than one epithelial counterreceptor exists, further complicating efforts at ligand identification by standard techniques such as antibody inhibition assays. This possibility is supported by the observation that adhesion of ICAM-1-expressing epithelial cells to CD11b/CD18-coated surfaces is not inhibited by anti-ICAM-1 antibodies. Indeed, with the above scenario, it is likely that functional antibodies to important epithelial ligands will only lead to partial inhibitory effects.

In summary, this article highlights our current understanding of PMN interactions with intestinal epithelial cells. Clearly, much remains to be learned about the details of the passage of PMN from the microcirculation, across the interstitium, and finally through the epithelium. Viewing the process from the epithelium, it has been determined that CD11b/CD18-mediated adhesion is an initial event in PMN interactions with epithelial cells that is followed by CD47-mediated transepithelial migration. These adhesive interactions have many unique features distinguishing them from PMN interactions with vascular endothelium or matrix. Although the nature of the epithelial counterreceptors for migrating PMN remains to be defined, further studies will likely yield new receptors and provide insights into new therapeutic modalities aimed at inhibiting PMN transepithelial migration and attenuating deleterious sequelae of PMN interactions with epithelial tissues.

    ACKNOWLEDGEMENTS

I would like to acknowledge the work of many investigators that helped to form the basis of this Themes article and could not be referenced due to editorial guidelines. Special thanks to Jim Madara and other members of the Epithelial Cell Biology Unit at Brigham and Women's Hospital for their valued collaborations and support.

    FOOTNOTES

* First in a series of invited articles on Cell Adhesion and Migration.

Work in my laboratory is supported by National Institutes of Health Grants HL-54229 and HL-58467 and by a research grant from the Arthritis Foundation.

Address reprint requests to Dept. of Pathology and Laboratory Medicine, Emory Univ., Woodruff Memorial Research Bldg., Rm. 2309, Atlanta, GA 30322.

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AJP Gastroint Liver Physiol 273(4):G763-G768
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